CA2228404A1 - Method for characterization of the fine structure of protein binding sites - Google Patents

Abstract

The binding sites of binding proteins and their binding partners are characterized, at the individual amino acid level, by a combination of heavy hydrogen (tritium or deuterium) exchange labeling and sequential degradation (preferably, by carboxypeptidase) and analysis of labeled fragments under slowed exchange conditions. By first labeling the binding partner and labeling the binding site of the protein indirectly (by exchange at the binding surface), the binding site may be differentiated from allosterically protected segments of the binding protein.

Description

- -CA 02228404 l998-0l-30 W O97/41436 PCT~US96/06011 ~LG~. 0~ FOR ~rTERIZATION OF THE FINE
~ l~U~-LUKE OF PROTEIN BI ~ ING SITES

This application is a continuation-in-part of Serial No.
08/240,593, hereby incorporated by re~erence in its entirety.

BAC~GROUn~D OF ~ E lNV~llON

Field o~ the Invention The present invention relates to the characterization o~ the binding site involved in binding between a binding protein and a binding partner.

Background Art Limitations o~ Curren~ Met~ods of Characterizing Pro~eln Rin~;ng Sites.
Considerable experimental work ,and time are required to precisely characterize a binding site. ~n general, the techniques which are the easiest to use and which give the quickest answers, result in an inexact and only approximate idea o~ the nature o~ the critical structural ~eatures. Techniques in this category include the study o~ proteolytically generated ~ragments o~ the protein which retain binding ~unction;
~0 recombinant DNA techniques, in which proteins are constructed with altered amino acid sequence (site directed mutagenesis);
epitope scanning peptide studies (construction o~ a large number o~ small peptides representing subregions o~ the intact protein ~ollowed by study o~ the ability o~ the peptides to inhibit binding o~ the ligand to receptor); covalent crosslinking o~ the protein~to its binding partner in the area o~ the binding site, ~ollowed by ~ragmentation o~ the protein and identi~ication of crosslinked ~ragments; and af~inity labeling o~ regions o~ the receptor which are located near the ligand binding site o~ the receptor, ~ollowed by characterization o~ such "nearest neighbor"
peptides. (Reviewed in 1, 2).
These techniques work best ~or the det~rm, n~ tion o~ the ~ structure o~ binding subregions which are simple in nature, as ~ when a single short contiguous stretch o~ polypeptide wi~hin a protein is responsible ~or most o~ the binding activity.

CA 02228404 l998-0l-30 WO 97/41436 PCTrUS96/06011 However, ~or many protein-binding partner systems o~ current interest, the structures responsible ~or binding on both receptor and ligand or antibody are created by the complex interaction o~
multiple non-contiguous peptide sequences. The complexities o~
these interactions may con~ound conventional analytical techniques, as binding ~unction is o~ten lost as soon as one o~
the 3-~ n~ional con~ormations of the several contributing polypeptide sequences is directly or indirectly perturbed.
The most de~initive techniques ~or the characterization o~
the structure o~ receptor binding sites have been NMR
spectroscopy and X-ray crystallography. While these techniques can ideally provide a precise characterization o~ the relevant structural ~eatures, they have major limitations, including inordinate amounts o~ time required ~or study, inability to study large proteins, and, ~or X-ray analysis, the need ~or protein-binding partner crystals (Re~. 3).
Applicant's technology overcomes these limitations and allows the rapid identi~ication o~ each o~ the speci~ic polypeptides and amino acids within a protein which constitute its protein ligand binding site or antibody binding subregion in virtually any protein-ligand system or protein antigen-antibody system, regardless o~ the complexity o~ the binding sites present or the size o~ the proteins involved. This technology is superior in speed and resolution to currently employed biochemical techniques.

Hydrogen (Proton) Exchange.
When a protein in its native ~olded state is incubated in bu~ers containing heavy hydrogen (tritium or deuterium) labeled water, heavy hydrogen in the bu ~er reversibly exchanges with normal hydrogen present in the protein at acidic positions (~or example, O-H, S-H, and N-H groups) with rates o~ exchange which are dependent on each exchangeable hydrogen's chemical environment, temperature, and most importantly, its accessibility to the tritiated water in the bu~er. (Re~s. 4, 5) Accessibility is determined in turn by both the sur~ace (solvent-exposed) - disposition of the hydrogen, and the degree to which it is hydrogen-bonded to other regions o~ the ~olded protein. Simply W O ~7/41436 PCT~US96/06011 stated, acidic hydrogen present on amino acid residues which are on the outside (bu~er-exposed) sur~ace o~ the protein and which are hydrogen-bonded to solvent water will exchange more rapidly with heavy hydrogen in the bu~er than will similar acidic hydrogen which are buried and hydrogen-bonded within the ~olded protein.
Hydrogen exchange reactions can be greatly accelerated by both acid and base-mediated catalysis, and the rate o~ exchange observed at any particular pH is the sum o~ both acid and base mediated mechanisms. For many acidic hydrogen, a pH o~ 2.7 results in an overall m;n~mllm rate o~ exchange (Re~. 6, pg.238, Figure 3, re~s. 7-11). While hydrogens in protein hydroxyl and amino groups exchange with tritium in bu~er at millisecond rates, the exchange rate o~ one particular acidic hydrogen, the peptide amide bond hydrogen, is considerably slower, having a hal~ e o~ exchange ~when ~reely hydrogen bonded to solvent water) o~ approximately 0.5 seconds at 0~C. pH 7, which is greatly slowed to a hal~ e o~ exchange o~ 70 minutes at 0~C
pH 2.7. _ -When peptide amide hydrogens are buried within a ~olded protein, or are hydrogen bonded to other parts o~ the protein, exchange hal~ lives with solvent hydrogens are o~ten considerably lengthened, at times being measured in hours to days. Hydrogen exchange at peptide amides is a ~ully reversible reaction, and rates o~ on-exchange (solvent heavy hydrogen replacing protein-bound normal hydrogen) are identical to rates o~ o~-exchange (hydrogen replacing protein-bound heavy hydrogen) i~ the state o~ a particular peptide amide within a protein, including its chemical environment and accessibility to solvent hydrogens, remains identical during on-exchange and o~-exchange conditions.
Hydrogen exchange is commonly measured by per~orming studies with proteins and aqueous bu~ers that are di~erentially tagged with pairs o~ the three isotopic ~orms o~ hydrogen (IH;Normal Hydrogen; 2H;Deuterium; 3H;Tritium). I~ the pair o~ normal hydrogen and tritium are employed, it is re~erred to as tritium exchange; if normal hydrogen and deuterium are employed, as - deuterium exchange. Di~erent physicochemical techni~ues are in general used to ~ollow the distribution o~ the two isotopes in PCT/u~ OC

deuterium versus tritium exchange.

Tri tium }~xchange Techni~ue~~
Tritium ~chAnge techniques (where the amount o~ the isotope is determined by radioactivity measurements) have been extensively used ~or the measurement o~ peptide amide exchange rates within an individual protein (reviewed in ~). The rates o~ exchange o~ other acidic protons ~OH, NH, SH) are so rapid that they cannot be ~ollowed in these techniques and all subsequent discussion re~ers exclusively to peptide amide proton exchange. In these studies, puri~ied proteins are on-exchanged by incubation in bu~ers cont~;n;ng tritiated water ~or varying periods o~ time, trans~erred to bu~ers ~ree o~ tritium, and the rate o~ o~-exchange o~ tritium determined. By analysis o~ the rates o~ tritium on- and o~-exchange, estimates o~ the numbers of peptide amide protons in the protein whose exchange rates ~all within particular exchange rate ranges can be made. These - studies do not allow a determination o~ the identity (location within the protein's primary amino acid sequence) o~ the exchanging amide hydrogens measured.
Extensions o~ these techniques have been used to detect the presence within proteins o~ peptide amides which experience allosterically-induced changes in their local chemical environment and to study pathways o~ protein ~olding (5, 12-14).
For these studies, tritium on-exchanged proteins are allowed to o~-exchange a~ter they have experienced either an allosteric change in shape, or have undergone time-dependent ~olding upon themselves, and the number o~ peptide amides which experience a chanqe in their exchange rate subsequent to the allosteric/~olding modi~ications determined. Changes in ~ch~nge rate indicate that alterations o~ the chemical environment o~
particular peptide amides have occurred which are relevant to proton exchange (solvent accessibility, hydrogen bonding etc.).
Peptide amldes whlch undergo an induced slowing in their exchange rate are re~erred to as "slowed amides" and i~ previously on-exchanged tritium is su~ficiently slowed in its o~-exchange ~rom such amides there results a "~unctional tritium labeling" o~
these amides. From these measurements, in~erences are made as _ _ _ _ W O 97/41436 - PCTAUS96/OGOll to the structural nature of the shape changes which occurred within the isolated protein. Again, determination o~ the identity of the particular peptide amides experiencing changes in their environment is n~t possible with these techniques.
Four groups of investigators have described technical extensions (collectively referred to as medium resolution tritium exchanqe~ which allow the locations of particular slowed, tritium labeled peptide amides within the primary sequence o~ small proteins to be localized to a particular proteolytic ~ragment, though not to a particular amino acid.
Rosa and Richards were the first to describe and utilize medium resolution tritium techniques in their studies o~ the folding of ribonuclease S protein ~ragments (15-17). However, the techniques described by Rosa and Richards were of marginal utility, primarily due to their failure to optimize certain critical experimental steps (reviewed in 6, pg 238, 244). No studies employing related techniques were published until the - work of Englander and co-workers in which extensive modi~ications and optimizations o~ the Rosa and Richards technique were ~irst described.
Englander's investigations utilizing.tritium exchange have ~ocused exclusively on the study of allosteric changes which take place in tetrameric hemoglobin (~ subunit and ~ subunit 16 kD in size each) upon deoxygenation (6,1~-21). In the Englander procedure, native hemoglobin (milligram quantities) in the oxygenated state is on-exchanged in tritiated water of relatively low specific activity (2-100 m~i/ml). The hemoglobin is then deoxygenated (inducing allosteric change), trans~erred to tritium-free ~uf~ers by gel permeation column chromatography, and then allowed to out-exchange for 10- 50 times the on-exchange time. On-exchanged tritium present on peptide amides which experience no change in exchange rate subsequent to the induced allosteric change in hemoglobin structure off-exchanges at rates identical to its on-exchange rates, and therefore is almost totally removed from the protein after the long off-exchange period. However, peptide amides which experience slowing of their exchange rate subsequent to the induced allosteric changes preferentially retain the tritium labe~. during the period of off-CA 02228404 l998-0l-30 W O97/41436 PCT~US96/06011 exchange.
To localize (in terms o~ hemoglobin's primary sequence-) the slowed amides bearing the residual tritium label, Englander then proteolytically ~ragments-the o~-exchanged hemoglobin with the protease pepsin, separates, isolates and identi~ies the various peptide fragments by reverse phase high pressure liquid chromatography (RP-HPLC), and determines which ~ragments bear the residual tritium label by scintillation counting. However, as the fragmentation of hemoglobin proceeds, each ~ragment's secondary and tertiary structure is lost and the unfolded peptide amides become ~reely accessible to H20 in the buffer. At physiologic pH (>6), any amide-bound ~ritium label would leave the un~olded fragments within seconds. Englander there~ore performs the ~ragmentation and ~P~C peptide isolation procedures under conditions which he believes m; n; m;ze peptide amide proton exchange, including cold temperature (4~C) and use o~ phosphate buf~ers at pH 2.7 (reviewed in 6). This techni~ue has been used successfully by Englander to coarsely identi~y and localize the peptidic regions o~ hemoglobin a! and ~ ~h~;rl~ which participate in deoxygenation-induced allosteric changes (18-21). The ability o~ the Englander techni~ue to localize tritium labeled amides, while an importa~t advance, r~m~n~ low; at the best, Englander reports that his technique localizes amide tritium label to hemoglobin peptides 14 amino acids or greater in size, without the ability to ~urther sublocalize the label.
In Englander's work, there is no appreciation that a suitably adapted tritium exchange techni~ue might be used to identi~y the peptide amides which reside in the contacting sur~ace of a protein receptor and its binding partner: his disclosures are concerned exclusively with the mapping of allosteric changes in hemoglobin. Furthermore, based on his optimization studies (6-11,13), Englander teaches and warns that a pH o~ 2.7 must be employed in both the proteolysis and HP~C
steps, necessitating the use of proteases which are ~unctional at these pH's (acid proteases). Un~ortunately, acid proteases are relatively nonspecific in their sites o~ proteolytic cleavage, leading to the production o~ a ~ery large number o~
di~erent peptide ~ragments and hence to considerable HPLC

W O 97/41436 PCT~S961060rl separation di~iculties. The constraint o~ per~orming the HPLC
separation step at pH 2. 7 gréatly limits the ability to optimize the chromatographic separation o~ multiple overlapping peptides by varying the pH at which HPLC is per~ormed. Englander tried to work around these problems, ~or the localiza~ion o~ hemoglobin peptides experiencing allosteric changes, by taking advantage o~
the ~act that some peptide bonds are somewhat more sensitive to pepsin than others. He there~ore limits the duration o~ exposure o~ the protein to pepsin to reduce the number o~ ~rayments. Even then the ~ragments were "di~icult to separate cleanly". They were also, of course, longer ~on average), and therefore the resolution was lower. He also tried to simpli~y the patterns by ~irst separating the alpha and beta rh~ ~ n~ o~ hemoglobin.
However, there was a tradeo~: increased tritium loss during the alpha-beta separation and the removal o~ the solvent, preparatory to proteolysis. Englander concludes, - "At present the total analysis o~ the HX (hydrogen exchange) behavior o~ a given protein by these methods i9 an immen,Ce task. In a large sense, the best strategies for undertaking such a task remain to be ~ormulated. Also, these e~orts would bene~it ~rom ~urther technical improvements, ~or example in HPLC
separation capability and perhaps especially in the development o~ additional acid proteases with properties adapted to t~e needs o~ these experiments"
(6).

Over the succeeding seven years since this observation was made, no advances have been disclosed which address these critical limitations o~ the medium resolution tritium exchange technique. It has been perceived that improvements to the HPLC
separation step were problematic due to the constraint o~ workiny at pH 2.7. The current limited success with small proteins has made it pointless to attempt similar studies o~ larger proteins where the problems o~ inadequate HPLC peptide separation at pH

2.7, and imprecision in the ability to sublocalize labeled amides would be greatly compounded. ~urthermore, most acid-reactive WO 97/41436 PCT~US~G/O~Ol1 proteases are in general no more speci~ic in their cleavage patterns than pepsin and e~orts to improve the technology by employing other acid reactive proteases other than pepsin have not signi~icantly improved the technique. Given these limitations o~ medium resolution tritium exchange art, no studies have been disclosed which utilize proteins with subunit size greater than 16 kilodaltons.
Allewell and co-workers have disclosed studies utilizing the Englander techniques to localize induced allosteric changes in the enzyme escherichia coli aspartate transcarbamylase (22,23).
Burz, et al. (22) is a brie~ disclosure in which the isolated R2 subunit o~ this enzyme is on-exchanged in tritiated bu~er o~
speci~ic activity 100 mCi/ml, allosteric change induced by the addition o~ ATP, and then the con~ormationally altered subunit o~f-exchanged. The enzyme ~2 subunit was then proteolytically cleaved with pepsin and analyzed ~or the amount o~ label present in certain ~ragments. Analysis employed techniques which rigidly ~ adhered to the rec~mm~n~tions o~ Englander, utilizing a single RP HPLC separation in a pH 2.8 bu~er.
The authors note di~iculty in separating the large number o~ peptides generated, even ~rom this small protein sub~ragment, given the constraints o~ the Englander methodology. They comment that "the principal limitation o~ this method at present is the separation with columns now available". ATP binding to the enzyme was shown to alter the rate o~ exchange o~ hydrogens within several relatively large peptidic ~ragments o~ the R2 subunit. In a subsequent more complete disclosure (23), the Allewell group discloses studies o~ the allosteric changes induced in the R2 subunit by both ATP and CTP. They disclose on-exchange o~ the R2 subunit in tritiated water-containing bu~er o~ speci~ic activity 22-45 mCi/ml, addition o~ ATP or CTP
~ollowed by o~ exchange o~ the tritium in normal water-cont~;n-ng bu~er. The analysis comprised digestion o~ the complex with pepsin, and separation o~ the peptide ~ragments by reverse phase HPLC in a pH 2.8 or pH 2.7 bu~er, all o~ which rigidly adheres to the teachings o~ Englander. Peptides were identi~ied by amino acid composition or by N-t~rm; n~ 1 analysis, and the radioactivity o~ each ~ragment was det~rm;n~d by rCT/US96/0601i scintillation counting. In both o~ these studies the localization of tritium label was limited to peptides which averaged 10-15 amino acids in size, without higher resolution being attempted.
Finally, Beasty, et al. ~24) have disclosed studies employing tritium exchange techniques to stud~ ~olding of the subunit of E. Coli tryptophan synthetase. The authors employed tritiated water of speclfic activity 20 mCi/ml, and fragmented the tritium labeled en~yme protein with trypsin at a pH 5.5, conditions under which the protein and the large fragments generated retained su~icient ~olded structure as to protect amide hydrogens from off exchange during proteolysis and HPLC
analysis. Under these conditions, the authors were able to produce only 3 protein fragments, the smallest being 70 amino 1~ acids in size. The authors made no further attempt to sublocalize the label by further digestion and/or HPLC analysis.
Indeed, under the experimental conditions they employed (they - performed ail steps at 12~C instead of 4~C, and per~ormed proteolysis at p~ 5.5 instead o~ pH in the range of 2-3), it would have been impossible to further sublocalize the labeled amides by tritium exchange, as label would have been ;m~;ately lost ~off~ h~nged) by the unfolding of subsequently generated proteolytic ~ragments at pH 5.5 if they were less than 10-30 amino acids in size.
In summary, the above disclosures are restricted to studies o~ medium resolution tritium exchange of: 1) The re-~olding on itsel~ of different parts of an individual protein (tryptophan synthetase ~ subunit) (24), 2) The re-~olding onto itsel~ o~ two ~ragments proteolytically generated from the same protein (ribonuclease-S) (1~-17); 3) The changes in shape (allosteric change) which an individual protein (hemoglobin) underwent subsequent to removal of oxygen (hemoglobin) (4-6,12-14,18-21);
and 4) The allosteric changes in a protein after the addition of known allosteric change inducers ~aspartate transcarbamylase) (22,23).
Because tritium exchange art was llmited in its ability to study large proteins, none of these or other investigators disclosed or proposed that tritium exchange techniques could be adapted to e~ectively study contact sur~aces between two di~erent, larqe proteins (subunits ~16 kD in size) or -that peptide amides ~unctionally labeled with tritium in large protein-binding partner ~interactions could e~fectively be localized precisely at the amino acid sequence level.
Fromageot, et al., U.S. Patent 3,828,102 (25) discloses using hydrogen exchange to tritium label a protein and its binding partner. The protein-binding partner complex is ~ormed be~ore allowing on-exchange to occur and thus the binding site is not selectively labeled. In the present invention the protein is on-exchanged be~ore its interaction with binding partner and subsequent o~-exchange, and thus, the peptide amides which reside in the interactions sur~ace speci~ically retain label while other sites do not.
Benson, U.S. Patents 3,560,158 and 3,623,840 (26) disclose using hydrogen exchange to tritiate compounds ~or analytical purposes. These re~erences di~er ~rom the invention by not providing any mechanism ~or distinguishing between any potential binding site and the rest o~ the molecule.

2 0 Deuteriulrl Exchange Techniques Fesik, et al (27) discloses measuring by NM~ the hydrogen (deuterium) exchanye of a peptide be~ore and a~ter it is bound to a protein. From this data, the interactions o~ various hydrogens in the peptide with the binding site o~ the protein are analyzed.
Patterson, et al. (28) and Mayne, et al. (29) disclose NMR
mapping o~ an antibody binding site on a protein (cytochrome-C) using deuterium exchange. This relatively small protein, with a solved NMR structure, is ~irst complexed to anti-cytochrome-C
monoclonal antibody, and the pre~ormed complex then incubated in deuterated water-cont~;n~ng bu~ers and NMR spectra obtained at several time intervals. The NMR spectra o~ the antigen-antibody complex is examined ~or the presence o~ peptide amides which experience slowed hydrogen ~ch~nge with solvent deuterium as compared to their rate o~ exchange in uncomplexed native - cytochrome-C. Benjamin, et al. (30) employ an identical NMR-deuterium techni~ue to study the interactlon o~ hen egg CA 02228404 l998-0l-30 W O97/41436 PCTrUS96/06011 lysosozyme (HEL) with HEL-speci~ic monoclonal antibodies. While both this NMR-deuterium technique, and medium resolution tritium exchange rely on the ph~nomenon o~ proton exchange at peptide amides, they utilize radically di~erent methodologies to measure and localize the ~xch~nging amides. Furthermore, study of proteins by the NMR techni~ue is not possible unless the protein is small (less than 30 kD~, large amounts o~ the protein are available ~or the study, and computationally intensive resonance assignment work is completed.
Recently, others (45-50) have disclosed techniques in which exchange-deuterated proteins are incubated with binding partner, o~-exchanged, the complex ~ragmented with pepsin, and deuterium-bearing peptides identi~ied by single stage fast atom bombardment IFab) or electrospray mass spectroscopy ~MS). In these studies, no attempt has been made to sublocalize peptide-bound deuterium within the proleolytically or otherwise generated peptide ~ragments.
-Sl~~RY OF THE lNVI~iNllON
The present invention provides methods ~or the ~unctional labeling o~ speci~ic amino acid residues that participate inbinding protein-binding partner interactions. It is particularly suitable ~or the study o~ the binding protein-binding partner subregions o~ large (~30 KD) proteins, even in small quantities.
In one embodiment, the label is tritium and the amount of label on a ~ragment or sub~ragment is determined by measuring its radioactivity. In a second embodiment, the label is deuterium and the amount o~ label on a ~ragment or subfragment is determined by mass spectrometry. The term "heavy hydrogen" is used herein to re~er generically to either tritium or deuterium.
In addition, re~erences to tritium apply mutatis mutandis to deuterium except when clearly excluded.
In essence, the binding protein is ~irst tritiated or deuterated under conditions wherein native hydrogens are replaced by the tritium or deuterium label (this is the "on-exchange"
step). Then the binding partner is allowed to interact with - labeled protein. The binding partner occludes the binding site and protects the tritium or deuterium labels o~ that site ~rom PCr/U,.~ Oll a subsequent "o~-exchange". ~hus, a~ter the "o~-exchange", only the bindinq site residues are labeled. Since the binding site is normally only a small portion o~ the molecules, a higher signal-to-background rati~ is obtained with this approach than with Englander's more conventional procedure.
In order to actually identify the labeled residues, one must ~irst dissociate the complex under slow hydrogen isotope exchange (H3/HIor H2/HI) conditions, since otherwise the labels would leave the binding site as soon as the ligand was removed. The binding protein is then ~ragmented (e.g., with an endoprotease such as pepsin), still under slow hydrogen exchange conditions, to obtain fragments. Those fragments which bear label presumably include binding site residues. At this point, the resolution o~ the binding site is no better than the fragment size.
A finer localization of the labels is achieved by analysis o~ sub~ragments generated by controlled, stepwise, degradation of each isolated, labeled peptide fragment under slowed exchange conditions. For the purpose o~ the present invention, a peptide fragment is said to be "progressively", "stepwise" or ~'seguentially~ degraded i~ a series o~ ~ragments are obtained which are typical of that which would be achieved by an ideal exopeptidase, that is, at each step, only an end amino acid is removed. Thus, if the n amino acids o~ a peptide were labled A~
to ~ (the numbering starting at whichever end the degradation begins), the sub~ragments would be A2...~, A3...~,..., ~-l-~, and ~lnally ~ The signals produced by the successive subfragments are correlated in order to determine which amino acids of the fragment in question were labeled.
This procedure was not used in any o~ the cited references to further localize the labeling sites, though improved resolution was certainly a goal o~ the art. The closest the art comes is Englander's general suggestions of ~urther fragmentations with another "acld protease".
The progressive degradation is pre~erably achieved by an enzyme, and more pre~erably by a carboxypeptidase. The need to employ an acidic pH at the time o~ degradation to m; n 1 m~ ze tritium losses discourages use o~ carboxypeptidases which are substantially inactivated by the required acidic bu~ers.

W O97/41436 PCT~US96/06011 13 However, carboxypeptidase-P, carboxypeptidase Y, and several other acid - reacti~e (i.e.. enzYmatically active under-acid conditions) car~oxypeptidases are suitable ~or proteolysis of peptides under acidic conditions, even at pH 2.7. Progressive sub~ragmentation of puri~ied tritium label-bearing peptides is per~ormed with acid-reactive carboxy~eptidases under conditions that produce a complete set o~ amide-labeled daughter peptides each shorter than the preceding one by a single carboxy-term;n~l amino acid. HPLC analysis of the several members o~ this set o~
progressively truncated peptides allows the reliable assignment o~ label to particular amide positions within the parent peptide.
Alternatively, the present invention contemplates C-term~n~l chemical degradation techniques that can be per~ormed under "slow hydrogen exchange conditions" e.g., by penta~luoropropionic acid anhydride. The sensitivity o~ the technique may be improved by the use o~ reference peptide sub~raqments as HPLC mobility markers.
- In general, the art has given insu~icient consideration to the problems of denaturing the binding protein su~iciently to ~acilitate proteolysis under slow hydrogen exchange conditions.
Pepsin, for example, is much less active at 0~C. than at room temperature. While pepsin is able to extensively digest hemoglobin that has been denatured by acidic pH at O~C, certain other binding proteins, such as hen egg lysozyme, are much more resistant to denaturation by slow H-exchange conditions, and hence to subsequent pepsin digestion. As a result, many ~ewer and longer ~ragments are generated. This complicates the analysis.
In a pre~erred embodiment, the labeled binding protein is exposed, be~ore ~ragmentation, to denaturing conditions compatible with slow hydrogen ~h~nge and su~ficiently strong to denature the protein enough to render it adequately susceptible to the intended proteolytic treatment. I~ these denaturing conditions would also denature the protease, then, prior to proteolysis, the denatured protein is switched to less denatured conditions (still compatible with slow H-exchange) su~iciently denaturing to maintain the protein in a protease-susceptible state but substantially less harm~ul to the protease =

W O 97/41436 PCT/U~6/~

in ~uestion.
Pre~erably, the initial denaturant is guanidine thiocyanate, and the less denaturing condition is obtained by dilutïon with guanidine HCl.
Disul~ide bonds, i~ present in the binding protein to be digested, can also inter~ere with analysis. Disul~ide bonds can hold the protein ln a ~olded state where only a relatively small number o~ peptide bonds are exposed to proteolytic attack. Even i~ some peptide bonds are cleaved, ~ailing to disrupt the disul~ide bonds would reduce resolution o~ the peptide ~ragments still ~oined to each other by the disul~ide bond; instead o~
being separated, they would remain together. This would reduce the resolution by at least a ~actor o~ two (possibly more, depending on the relationship o~ disul~ide bond topology to peptide cleavage sites). I~ the disul~ide bonds are not disrupted, ~urther sublocalization o~ the tritium-labeled amides within each o~ the disul~ide-joined peptides would be very di~icult, as amino acid removal would occur, at dif~erent times and at di~erent rates, at each C-t~m; n~ 1 0~ the disul~ide linked segments.
The applicant has discovered that water soluble phosphines may be used to disrupt a protein's disul~ide bonds under "slow hydrogen exchange" conditions. This allows much more e~ective ~ragmentation o~ large proteins which contain disul~ide bonds without causing tritium label to be lost ~rom the protein or its proteolytic fragments (as would be the case with conventional disul~ide reduction techniques which must be per~ormed at pH's which are very un~avorable ~or preservation o~ tritium label).
In another embodiment, peptide amides on the binding protein's sur~ace are indirectly labeled by trans~er o~ tritium or deuterium that has been previously attached by hydrogen exchange to the interaction sur~ace of the binding partner. This procedure will ~unctionally label receptor protein amides i~ they are slowed by complex ~ormation and are also in intimate contact with the binding partner, in the complexed state. Amides that are distant ~rom the interaction sur~ace but slowed in exchange because o~ complex ~ormation-induced allosteric changes in the protein will not ~e labeled.

W O 97/41436 PCTrUS96/~6011 BRIEF DESCRIPTION OF THF DRU~WINGS
Fiqure 1. Analysis o~ tritium associated with hemoglobin (-Hgb) ~ragments produced by pepsin digestion of tritium-e~changed hemoglobin + monoclonal,~ antibody ~ollowed by HPLC in PO4 bu~fered solvents, pH 2.7. Panel A: Absorbance (214 nM) tracing o~ unlabeled proteolyzed Hgb. Panel B: Hgb on-exchanged for 4 hours, shi~ted to pH 2.7 and then proteolyzed without o~
exchange. Panel C: Hgb on-exchanged for 4 hours, mixed with monoclonal ~6 and then o~f-exchanged ~or 40 hours before proteolysis at pH 2.7. Panel D: Hgb on-exchanged ~or 4 hours and then o~-exchanged ~or 40 hours be~ore proteolysis at pH 2.7.

Fiqure 2. Second ~;m~n~ion separation (HPLC with 0.1 Tri~luroracetic Acid (TFA) containing solvents) at 0~C of tritium-bearing rpHPLC ~raction from ~irst ~;m~n~ion separation, Figure 1, panel C.

Fiqure 3. Panels A to C. Identi~ication o~ hemoglobin peptides ~unctionally labeled by interaction with monoclonal ~121.
Similar to Figure 1 but employing monoclonal ~121 in place of monoclonal ~6.

Fiqure 4. Panels A to D. Identi~ication o~ hemoglobin peptides ~unctionally labeled by interaction with haptoglobin. Similar to Figure 1, but employing haptoglobin in place o~ antibody.

Fiqure 5. Structure o~ hemoglobin with peptidic regions highlighted., Panel A: ~ monoclonal interaction peptides; Panel B: ~121 monoclonal interaction peptides.

Fiqure 6 Carboxypeptidase-P digestion of ~1-14 peptide.
Tritium-exchange-labeled synthetic ~1-14 peptide was digested (0~C) with carbo~ypeptidase-P (CP-P) using a range o~ enzyme concentrations and digestion times (indicated at ~ar left margin). HPLC analysis as then per~ormed as in Figure 1, but with simultaneous measurement of O.D.214 (le~t panels) and - radioactivity (right panels) o~ column ef~luent. The positions o~ the several generated C-te~m; n~ 1 truncated peptide ~ragments CA 02228404 l998-0l-30 WO 97/41436 PCT~US96/06011 are indicated (numbers 3 through 9). Progressive generation o~
~ragments is observed.

Fiqure 7. Reduction o~ aisul~ide bonds at pH 2.7. Tritium-exchange-labeled ~ peptide (2~g at 0~C, pH 2.7) was supplemented with the peptide endothelin (4~g), which contains two disul~ide bonds (35), and the mixture incubated without (A) or with (B-E) 50 mM Tris (2-carboxyethyl) phosphine (TCEP) ~or varying times at 0~C (A,C-E), or 2 minutes at 22~C (B). The mixtures were then subjected to HPLC as in ~igure 7. The percent o~ endothelin that remained unreduced under each condition is indicated (le~t panels) as is the ~raction o~ tritium label that remained attached to the ~1-14 peptide (right panels). Fi~ty percent reduction o~ endothelium disul~ides is accomplished at pH 2.7 with an insigni~icant loss o~ peptide amide-bound tritium ~rom the ~1-14 peptide. "R" indicates the positions o~ reduced ~orms o~ endothelin.
.

DETATT~n DESCRIPTION OF THE PREFERRED E~IBODI~ENTS
Biochemical Binding, Generall~.
Many biological processes are mediated by noncovalent binding interactions between a protein and another molecule, its binding partner. The identi~ication o~ the structural ~eatures of the two binding molecules which immediately contribute to those interactions would be use~ul in designing drugs which alter these processes.
The molecules which pre~erentially bind each other may be re~erred to as members o~ a "speci~ic binding pair" Such pairs include an antibody and its antigen, a lectin and a carbohydrate which it binds, an enzyme and its substrate, and a hormone and its cellular receptor. In some texts, the terms "receptor'l and "ligand" are used to identi~y a pair o~ binding molecules.
Usually, the term "receptor" is assigned to a member o~ a specific binding pair which is o~ a class o~ molecules known ~or its binding activity, e.g., antibodies. The term "receptor'~ is also pre~erentially con~erred on the member o~ the pair which is -35 larger in size, e.g., on avidin in the case o~ the avidin-biotin pair. However, the identi~ication o~ receptor and ligand is W O 97/41436 PCT~US96/06011 ultimately arbitrary, and the term "ligand" may be used to re~er to a molecule which others would call a "receptor". The term "anti-ligand" is sometimes used in place o~ "receptor".
While binding interactions may occur between any pair o~
molecules, e.g., two strands o~ DNA, the present speci~ication is primarily concerned with interactions in which at least one o~ the molecules is a protein. Hence, it is convenient to speak o~ a "binding protein" and its "binding partner". The term ~protein" is used herein in a broad sense which includes, mutatis mutandis, polypeptides and oligopeptides, and derivatives thereo~, such as glycoproteins, lipoproteins, and phosphoproteins, and metalbproteins. The essential re~uirement is that the "binding protein" ~eature one or more peptide ~ -NHCO-) bonds, as the amide hydrogen o~ the peptide bond (as well as in the side rh;?;nq o~ certain amino acids) has certain properties which lends itself to analysis by proton exchange.
The binding protein may be identical to a naturally occurring protein, or it may be a binding ~ragment or other mutant o~ such a protein. The ~ragment or mutant may have the same or di~erent binding characteristic relative to the parental protein.
Integral membrane proteins are o~ particular interest, as they are di~icult to crystallize ~or study by X-ray di~raction.
Proteins too large to study by NMR methods, e.g., those larger than about 50 kDa, are also o~ special interest, particularly i~
they cannot be characterized as a composite o~ two or more separately analyzable dom~; n.q . Examples o~ suitable proteins include integrins (which are large integral membrane proteins), cell sur~ace receptors ~or growth ~actors (including cytokine receptors), '1seven-spanners", selectin, and cell surface receptors o~ the immunoglobin super~amily (e.g., ICAM-1).
The method o~ the present invention is especially use~ul ~or studying proteins with discontinuous epitopes, such as certain - antibodies, including certain clinically important autoimmune antibodies.
A "binding site" is a point o~ contact between a binding - sur~ace ("paratope") o~ the binding protein and a complementary sur~ace ("epitope") o~ the bindiny partner. (When the binding _ W 097/41436 PCT/U~55/~Oll partner i8 a protein, the designation o~ "paratope" and "epitope"
is essentially arbitrary. However, in the case o~ antibody-antigen interactions, it is conventional to re~er to the antigen binding site o~ the antibody as the "paratope" and the target site on the antigen as the l'epitope".) A speci~ic binding pair may have more than one binding site, and the term "pair" is used loosely, as the binding protein may bind two or more binding partners (as in the case o~ a divalent antibody). Moreover, other molecules, e.g., allosteric e~ectors, may alter the con~ormation o~ a member o~ the "pair" and thereby modulate the binding. The term ~Ipair~ is intended to encompass these more complex interactions.

Slowed Hydroqen Exchanqe Conditions The present invention contemplates labeling the binding site o~ a binding protein (or binding partner) with a heavy hydroge~
isotope, and dete~m; n; ng the location o~ the labels under slowed hydrogen ~h~nge conditions. "Slowed hydrogen exchange conditions~' are hereby defined as conditions wherein the rate o~
e~change o~ normal hydrogen ~or heavy hydrogen at amide hydrogens ~reely exposed to solvent is reduced substantially, i.e., enough to allow su~icient time to determine, by the methods described herein, the precise amide hydrogen positions which had been labeled with heavy hydrogen. The H-exchange rate is a ~unction o~ temperature, pH and solvent The rate is decreased three ~old ~or each 10~C drop in temperature. Hence use o~ temperatures close to 0~C is pre~erred. In water, the m;n;mllm H-exchange rate is at a pH o~ 2-3. As conditions diverge ~rom the optimum pH, the H e~change rate increases, typically by 10-fold per pH unit increase or decrease away ~rom the m~n~mllm. Use o~ high concentrations o~ a polar, organic cosolvent shi~ts the pH min to higher pH, potentially as high as pH 6 and perhaps, with the right solvent, even higher.
At pH 2.7 and 0~., the typical hal~ life o~ a tritium label at an amide position ~reely exposed to solvent water is about 70 minutes. Pre~erably, the slowed conditions o~ the present - inventions result in a hal~-life o~ at least 10 minutes, more pre~erably at least 60 minutes.

W O 97/41436 PCTnUS96/06011 Tritium Exchange Embodiments In one embodiment, the present invention contemplates- the ~ollowing procedure ~or characterization o~ a binding s~ite:
A. The ph~n~m~non o~ hydroyen (tritium) exchange is used to substitute a radioactive probe (tritium) ~or each o~ the amide hydrogens on the amino acids which make up the sur~ace o~ the receptor protein, including the sur~ace o~ the receptorls ligand binding site. This labelling is accomplished under essentially physiologic conditions by incubating the receptor protein in solutions containing tritiated water. (Pre~erably, the water is o~ high specific activity.) B. Protein ligand (binding partner) is then added to the on-exchanged (tritiated) receptor protein and allowed to bind to its speci~ic site on the receptor. Once the ligand has bound to the receptor, hydrogens on the amino acids which make up the surface o~ the receptor's binding site are no longer capable o~
e~iciently interacting with the surrounding aqueous bu~er, and ~urther hydrogen exchange is markedly inhibited.
C. The tritiated receptor-ligand complex is then trans~erred to physiologic bu~ers ~ree o~ tritium. Tritium label on the receptor-ligand complex is allowed to exchange o~ the receptor.
However, binding complex-dependent hydrogen-bonding between the protein and binding partner and limited solvent accessibility to the protein-binding partner inter~ace in the complex are selective impediments to the o~-exchange o~ peptide amide tritium label sandwiched between the protein and binding partner.
A~ter the removal (o~-exchange) o~ tritium ~rom other regions o~ the protein-binding partner complex is substantially ~inished, the result is the pre~erential retention o~ tritium label at the amides ~or which hydrogen exchange is slowed by virtue of protein-binding partner interactions, typically amides proximate to amino acids which make up the sur~ace o~ the receptor's ligand binding site. Optionally, the complex may be subjected to - limited proteolytic digestion, denaturation and/or disul~ide reduction while o~ exchange is proceeding, as long as the integrity o~ the binding protein: bindiny partner interaction is - not substantially perturbed by such maneuvers.
D. The speci~ic peptide bond amides which bear the r~m~;n;ng CA 02228404 l998-0l-30 W O97/41436 - PCT~US9G/06011 tritium are then identi~ied. This is done by:
(1) shi~ting the labeled receptor-ligand complex to conditions (e.g., 0-4~C, pH 2 7) which dissociate the complex and at the same time slow down~amide hydrogen exchange.
(2) subjecting the receptor to proteolysis ~ollowed by reverse phase (~P) high pressure liquid chromatographic (EPLC) separation (pre~erably 2-~lm~n~ional) o~ the resulting receptor fragments under continued slow proton exchange conditions.
Receptor ~ragments bearing tritium label are identi~ied, isolated, and characterized as to their amino acid sequence, and there~ore their location within the primary amino acid sequence o~ the intact receptor.
Preparation o~ the binding protein ~or proteolytic analysis may involve:
(a) trimming o~ o~ portions o~ the protein not required ~or complex ~ormation;
(b) disruption o~ disul~ide bonds which could complicate the analysis o~ the ~ragments (see section 5A); and/or (c) denaturation o~ the protein to render it more susceptible to proteolytic attack (see section 5B).
Step (a) may be per~ormed before or a~ter switching to slow hydrogen exchange conditions, since it does not cause dissociation o~ the contacting sur~aces. Steps (b) and (c) are more likely cause such dissociation and there~ore will more o~ten need to be per~ormed under slow exchange conditions.

(3) determ; n; ng the location o~ tritium label within each peptide by sub~ragmenting the labeled peptides (e.g., with acid-reactive carboxypeptidases or tritium-exchange-compatible chemical methods) under slow proton exchange conditions and characterizing the labelled sub~ragments. For example, the identity o~ each o~ the sub~ragments may be determined by amino acid analysis, peptide sequencing, or by comparison of their mobility with synthetic HPLC mobility marker peptides, and the amount o~ tritium label attached to each sub~ragment determined by scintillation counting. As each carboxy-t~rm; n~ 1 amino acid o~ the ~unctionally labeled peptide is sequentially cleaved o~
- by the carboxypeptidase, the nitrogen which ~ormed the slowly-exchanging peptide amide in the intact peptide bond is converted CA 02228404 l998-0l-30 W O 97/41436 PCT/U',-'~6011 21 to a rapidly exchanging secondary amine, and any tritium label at that nitrogen is lost ~rom the peptide within seconds, whereas all other amide bond tritium r~m~;nS in place. A stepdown in radioactivity ~rom one sub~ragment to the next smaller one indicates that the amide just altered had been labeled with tritium.
In this m~nn~r, the precise location, within the protein, o~ each peptide amide that is ~unctionally labeled with tritium by virtue o~ its interaction with binding partner is determined.
In~erentially, in this m~nn~r, the precise amino acids which make up the sur~ace o~ the receptor's binding site are then known.
Studies may be per~ormed to quanti~y the exchange rates o~ each of the labeled amides identi~ied above both be~ore and a~ter complex ~ormation with binding partner. This allows calculation o~ the maqnitude o~ exchanqe slowina experienced by each o~ these amides consequent to complex ~ormation, and allows optimization o~ on and o~ exchange times.
E. Parallel studies may be per~ormed in which the cognate bindinq partner is on-exchanged with tritium, complexed with receptor protein, o~-exchanged as a binding partner-protein complex and slowed amides in the bindinq partner identi~ied as above. This procedure results in the identi~ication o~ the subregions o~ the binding partner which interact with the protein.
F. The knowledge o~ the identity o~ the precise contact peptides in both receptor and ligand may be combined with additional structural in~ormation provided by the invention (identi~ication o~ peptide amides o~ the protein and binding partner which are likely to directly form hydrogen bonds between protein and binding partner upon complex ~ormation) to produce models ~or the complementary 3-~n~ional structures o~ the receptor and ligand interaction sur~aces. These models may then be used as the basis o~ the design and production o~ appropriate peptide and peptidomimetic drugs.
The individual steps o~ this procedure will now be considered in greater detail.
-1. On-Exchanqe CA 02228404 l998-0l-30 W O97/41436 PCT~US96tO6011 The protein under study is incubated in buf~er supplemented with tritiated water (3H2O), pre~erably of high specific activity.
This results in the time dependent reversible incorporation of tritium label into every peptide amide on the surface of the protein, including its ~potential) ligand binding subregion, through the mechanism of proton exchange Any physiologic buf~er appropriate for the interaction of the protein with its binding partner may be utilized (with no constraints imposed on buf~er pH or temperature). Suitable buffers include phosphate bu~ered saline, 0.15 mM NaCl, 10 mM
PO4, pH 7.4 PBS. The use of small incubation volumes (0.1-10 ,ul) containing high concentrations of receptor protein (10-100 mg/ml) is preferred.
The necessary level of tritiation (a~d hence the concentration of tritium in the buffer) is dependent on the totol amount of protein available ~or analysis. For analysis of lmg protein, at least 10 Ci/ml is desirable; for O.lmg, 100 Ci/ml, and i~or .Olmg, 1000 Ci/ml. (Pure tritiated ~I2O is about 2500 Ci/ml.) For most applications, the tritiated water will be 50-500 Ci/ml. Without the use o~ these high specific activities,studies of proteins which are available in limited quantity would be much more difficult. (Even higher speci~ic activity (e.g., 500-1,500 Ci/ml) may be used in the invention, but radiation safety considerations necessitate performance of such on- and o~f-exchange procedures in specialized ~acilities, such as are available in the tritium laboratory provided by the National Tritium ~acility, Lawrence Berkeley Laboratories, University of California, Berkeley.) It should be noted that with customary levels of tritium, only a small percentage o~ the binding protein molecules will be tritiated at any given exposed position. All that is required is that substantially each of the exposed amide hydrogen atoms be replaced in a detectable (by radiation counting) number of the binding protein molecules.
It is not necessary that the tritium exchange analysis rely on only a single choice o~ "on-exchange" time. Rather, the - skilled worker may carry out the experiment using a range of on-exchange times, preferably spanning several orders o~ magnitude W O 97/41436 PCT~US96/06~11 (seconds to days) to allow selection o~ on-exchange times which allow e~icient labeling o~ the various peptide amides present in the protein, which will become slowed in their ~rh~~nge rate consequent to the interaction o~ the protein to its binding partner, and at the same time m;n;m;ze background labeling o~
other amide positions a~ter o~f-~h~nge is completed (see section 10 below).

2. Receptor-Bindinq Partner Complex Formation A~ter a suitable period o~ tritium on-exchange, the protein~ 9 binding partner is added to the tritiated protein-bu~er solution and the two allowed to ~orm a binding complex.
The binding partner is pre~erably added in ~uantities su~icient to produce saturation ~inding to the protein (usually e~uimolar amounts) and at high concentrations (e.g., 10-100 mg/ml) to maximize the rate and extent o~ binding To m;n;mlze tritium labeling o~ the added binding partner by proton exchange (important when u~ilizing short on-exchange times), 3H20 in the bu~er is pre~erably diluted with tritium-~ree bu~er (10-1000 ~old dilution) within 0-100 seconds o~ binding partner addition.
Additional manipulations detailed below may be used at this step to ~urther m~n;m; ze incorporation o~ tritium label into the binding partner.

3 . Off -Exchanqe The tritiated protein-binding partner complex is then trans~erred to physiologic bu~ers identical to those employed during on-exchange, but which are substantially ~ree o~ tritium.
Tritium label on the protein then exchanges o~ the protein at rates identical to its on-exchange rate everywhere except at amides which have been slowed in their exchange rate by virtue 3 0 o~ the interaction o~ protein with binding partner. With su~icient o~-exchange time, the result is the speci~ic retention o~ tritium label at each of the peptide amide bonds which occur between the amino acids which make up the sur~ace o~
the protein's binding site ~or the binding partner. We re~er to -35 this process as a complex ~ormation-dependent ~unctional labelinq o~ the protein with tritium. At least 90~, more pre~erably, at W O 97/41436 PCT~US96/06011 least 99~, o~ on-exchanged tritium label at other sites is o~f-exchanged ~rom the protein.
In general, o~-~rh~nge is allowed to proceed ~or 5 to 50 times, more pre~erably about 10 times the on-exchange period, as this allows o~-exchange ~rom the protein o~ greater than 99~ o~
the on-exchanged tritium label which has not experienced a slowing o~ exchange rate subse~uent to the protein's interaction with binding partner. Prel; m~ n~y studies may be per~ormed with the protein and binding partner to determine the on and o~
exchange times which optimize the signal (tritium r~m~;n;ng in ~unctionally labeled amides) to noise (tritium r~m~;n;ng in background amides) ratio (see section 8).
In pre~erred embodiments, the o~-exchange procedure may be per~ormed with the use o~ Sephadex G-25 spin columns prepared and utilized as described in Example 1 (below), by G25 column chromatography as described by Englander (6,19) or by use o~
per~usive HPLC supports that allow rapid separation o~
peptide/protein ~rom solvent (Poros~ columns, PerSeptive Biosystems, Boston, MA). Use o~ the G25 spin columns allows the separation o~ the complex ~rom greater than 99.9~ o~ bu~er tritium. Residual bu~er tritium and tritium o~-exchanged ~rom the complex may optionally be ~urther removed by dialysis o~ the complex against tritium ~ree bu~er during o~ exchange.
Alternatively, complex ~ormation and o~-exchange can be accomplished by ~irst reacting the on-exchanged protein-bu~er mixture with binding partner which has been covalently attached to a solid support (e.g. binding-partner-Sepharose), allowing the on-~h~nged protein to complex to the solid-phase binding partner, ~ollowed by washing o~ the sepharose-binding partner-protein conjugate with tritium ~ree bu~er~ Alternatively,soluble protein-binding partner complexes may be ~ormed as above, and captured with a solid phase adsorbent that can bind to either the protein or binding partner component o~ the complex (e.g.
Sepharose with covalently attached antibodies speci~ic ~or protein or binding partner).
Most protein-ligand binding interactions that will be probed - with this technique are reversible reactions: binding partner will dissociate ~rom and rebind to the protein during the o~-W O 97/41436 - PCT/Ub~ 011 exchange period, and during the brie~ intervals where the ~rotein's binding site is unoccupied with binding partner, proton o~-exchange proceeds at the unprotected rate. It is there~ore important to m; n;m; ze the time that the binding site is unoccupied. In a pre~erred embodiment, this is accomplished by having both receptor and binding partner present at high concentration, e.g., at least mg/ml concentrations, up to 100 mg/ml concentrations each throughout the o~-exchange period, and per~orming the on and o~f exchange reactions at temperatures at or below room temperature, pre~erably 4~C.

4. Trimminq o~ the Bindinq Protein (optional) Prior to dissociation o~ the complex, e.g., during the o~-exchange period, which typically lasts hours to days, the complex may optionally be chemically or enzymatically treated to produce the smallest ~ragment o~ protein which is still capable o~
remaining tightly bound to the binding partner, and this residual "trimmed" complex isolated. Removal o~ portions o~ the protein not essential ~or continued complex ~ormation will decrease the number o~ background peptides generated during the subsequent acid proteolysis o~ the trimmed complex (Section 6). This pre-digestion and puri~ication can be per~ormed with a wide variety o~ proteases (e.g. trypsin, pronase, V-8 protease chymotrypsin proteinase-K) as well as certain chemical agents (e.g., cyanogen bromide, iodosobenzoic acid), and under virtually any conditions o~ induced partial protein denaturation (e.g. urea, guanidinium chloride sodium dodecyl sulfate, non-ionic detergents, reductants such as 2-mercaptoethanol, dithiothreitol), ionic strength, temperature, time and pH which do not substantially dissociate the contactinq sur~aces o~ the protein-bindinq partner complex.
Excessive digestion e~orts which result in dissociation o~ these sur~aces ~rom each other will cause a large ~raction o~
~unctional tritium label to be immediately o~-exchanged, as - greater than 50~ o~ peptide amides in the dissociating sur~aces will have exchange hal~-lives o~ less than 1 minute at pH
3~ approximately 7. The goal is to generate and isolate a ~ragment - o~ the protein, pre~erably 15-100 kD in size more pre~erably 15 kD, which r~m~; n.~ attached to the binding partner. O~ten "ligand W O 97/41436 PCT~US96/06011 stabilization" o~ proteins which are proteolysed while bound to binding partner allows the continued binding o~ the protein ~ragments to partner.
Prel;m;n~ry studies may be per~ormed with the o~-exchanged complex to det~rm; n~ conditions which result in a suitably trimmed protein-binding partner complex. In a pre~erred embodiment, the quantity o~ residual tritium ~unctionally bound to the intact o~-exchanged complex is first determined by measurement o~ tritium which migrates with the void volume (Mr 10 ~10,000 kD) on a G25 spin column (pH 7,a~). Aliquots of~ the complex are then subjected to varied ~ragmentation conditions, and the ~raction of tritium label which r~m~;nR attached to polypeptides under each digestion condition (migrates with G25 void volume) determin~d~ The proteolytic products o~ the most vigorous digestions which "release" less than 5~ o~ complex-associated tritium are (as per Section 5) adjusted to pH 2.7, O~C, subjected to RP-HPLC at pH 2.7, O~C, and peptides/protein ~ragments which bear label identified, isolated, and their molecular weights determined by SDS-PAGE. The labeled proteolytic products produced in these limited digests are likely to be large polypeptides, and therei~ore RP-HPLC supports suitable to the puri~ication o~ such peptides (C-4, phenyl columns) are utilized. Alternatively, when solid-phase adsorbents are used ~or complex ~ormation/o~-~ch~nge (step 3), proteolysis as above, now o~ the solid phase binding partner-protein complex, is allowed to proceed as extensively as possible without release ~rom the solid support o~ greater than 5~ functionally attached tritium. The predigested protein/complex is then released ~rom the immunoadsorbent with denaturants including a shi~t to pH 2.7, and the predigested protein further proteolysed with pepsin other acid reactive proteases.
A binding protein may also be trimmed earlier, e.g., be~ore '1on-exchange" or before complex formation, provided that the trimmed protein binds the partner su~iciently similarly to the original protein to be o~ interest.

- 5. Switch to Slow Amide Hydro~en Exchanqe Conditions The protein-binding partner complex (or predigested W O 97/41436 PCTAUS96/06~11 complex--see Step 4) is then shi~ted to conditions o~ temperature and pH which greatly 8~0w the halE liee o~ peptide amide hydrogen exchange, and essentially "~reeze" in place the protein binding site-retained tritium label. In a pre~erred embodiment, the complex is shiEted to 0~C, and pH 2. 7 conditions under which the half li~e o~ exchange o~ peptide amide label in ~ully denatured peptides is at least 70 minutes. The label will be su~f~iciently held in place under these conditions so that several rounds of proteolytic ~ragmentation, HP~C separation, and tritium quanti~ication can be per~ormed without unacceptable loss o~
label.
For some binding proteins, switching to slow hydrogen exchange conditions is su~icient to cause dissociation o~ the complex I~ not, a dissociating agent, such as a chaotropic agent may be added.
5A. Disruption o~ protein disul~ide bonds under acidic conditions: (optional) High resolution localization o~ tritium label-bearing amides requires the proteolytic generation o~ peptides less than approximately 1~-20 amino acids in size under conditions which allow the label to remain in place (e.g., 0~C, pH 2. 7). The ability o~ any protease to ~ragment a protein or peptide is limited by the accessibility o~ the protease to susceptible peptide bonds. While denaturants such a~ acidic pH, urea, detergents, and organic co-solvents can partially denature proteins and expose many otherwise structurally shielded peptide bonds, pre-existing disul~ide bonds within a protein can prevent su~icient denaturation with these agents alone. In conventional protein structural studies, disul~ides are usually cleaved by reduction with 2-mercaptoethanol, dithiothreitol, and other reductants which un~ortunately require a pH greater than 6 and elevated temperature ~or su~icient activity, and are there~ore not use~ul ~or the reduction o~ disul~ides at p~ 2. 7 or below.
For this reason, the tritium exchange art has not attempted any ~orm o~ disul~ide ~ond disruption, has ~or the most part been restricted to the study o~ proteins without intrinsic disul~ide - bonds, and has accepted the low resolution achievable without disul~ide ~ond disruption. The applicants have recognized and CA 02228404 l998-0l-30 6 PCTrUS96/060~1 demonstrated that acid-reactive phosphines such as Tris ~2-carboxyethyl) phosphine (TCEP) (31-36) can be used to disrupt disul~ides under the acidic pH and low temperature constraints required ~or tritium ~h~nge analysis (see Figure 7). We have established that these manipulations disrupt these associations and at the same time continue to produce a markedly slowed proton exchange rate ~or peptide amide protons.
5B. Protein Denaturation. (optional) In previous studies by Englander et al. and others, employing medium resolution tritium exchange, proteolytic ~ragmentation o~ tritium-labelled proteins under slowed-exchange conditions was accomplished by shi~ting the protein's pH to 2 . 7, adding high concentrations o~ liquid phase pepsin, ~ollowed by brie~ (10 min.) incubation at 0 C. With the proteins studied by Englander et al. simply shi~ting pH ~rom that o~ physiologic (7.0) to 2.7 was su~icient to render them suf~iciently denatured as to be susceptible to pepsin proteolysis at 0~C. Furthermore, these proteins, in general, did not contain disul~ide bonds that inter~ered with e~ective denaturation by such (acid) pH
conditions or contain disul~ide bonds within portions o~ the protein under study with the technique. The applicant has ~ound that other proteins (~or example hen egg lysozyme) are negligibly denatured and are not substantially susceptible to pepsin proteolysis when continuously incubated at comparable acidic pH
and depressed temperature (10-0 C). This is the consequence o~
the existence o~ a thermal barrier to denaturation for many proteins incubated in many denaturants; i.e., denaturation o~
proteins at lower temperatures (10-0~C) is o~ten ine~icient and a slow process, incompatible with the requirement o~ medium resolution tritium exchange techniques that manipulations be per~ormed rapidly, such that the attached tritium label is substantially retained at ~unctionally labelled amides o~ the binding protein.
The applicant has discovered that such proteins become extraordinarily susceptible to pepsin proteolysis at 0~C when they are treated with the sequential denaturation procedure - described below. Furthermore, the applicant has discovered that although TCEP can e~ect the reduction o~ disul~ide bonds in .
-W O 97/41436 PCT~US96/0601 proteins at 0~C and pHs in the range oi~ 2- 3, it is relatively ine~icient at doing so under these conditions and becomes much more e~icient at e~ecting reduction at a pH o~ 5.0 or greater.
Conditions can be arranged~to greatly increase the e~iciency o~
TCEP-mediated reduction while at the same time preserving 510w exchange conditions. This is accomplished by simultaneously denaturing the protein with guanidine thiocyanate, employing very high concentrations o~ TCEP and raising the pH o~ the solution to 5Ø While this pH would normally produces an unacceptable 100-~old increase (as compared to that at pH 2.7) in the rate o~
loss o~ tritium ~rom the labelled protein, the elevated pH-induced increase in the rate o~ tritium loss is substantially o~set by limiting the water content of the incubation mixture (and thereby markedly slowing the rate o~ tritium loss) when the protein is being reduced at pH 5.0, and the solution pH then is shi~ted back to pH 2.7 once reduction is complete. The result is e~ective reduction o~ proteins at a pH o~ 5 and 0~C with substantially complete rete~tion o~ tritium label on the binding protein.
The denatured (or denatured and reduced) protein solution is then passed over a pepsin-agarose column, resulting in e~icient and rapid ~ragmentation o~ the protein (in s 1 min.).
The ~ragments can be, and usually are, immediately analyzed on RP-HPLC without unnecessary contAm;n~tion o~ the peptide mixture with the enzyme pepsin or ~ragments o~ the enzyme pepsin Such contamination is problematic with the technique as taught by Englander, et al., as high concentrations o~ pepsin (o~ten equal in mass to the protein under study) are employed, to ~orce the proteolysis to occur su~iciently rapidly at 0~C.
While proteins are o~ten sub]ected to purpose~ul denaturation with agents other than a pH shift prior to digestion with pepsin, this has never been done at depressed temperatures (10- 0 C) be~ore, and the applicant has discovered that while - guanidine thiocyanate at the indicated concentrations is su~icient to suitably denature and render susceptible to pepsin proteolysis proteins at 10-0~C, several other strong denaturants, - including urea, HCl, sodium dodecyl sul~ate (SDS) and guanidine HCl, were, at least when used alone, unable to adequately W O 97/41436 PcT/u~' denature lysozyme at these low temperatures. However, the concentrations of guanidine thiocyanate required ~or such denaturation are incompatible with pepsin digestion; i.e., they denature the pepsin enzyme be~ore it can act on the denatured binding protein. When the guanidine thiocyanate is removed tat 10-0 C) ~rom the solution a~ter protein denaturation has been accomplished in an attempt to overcome this inhibition of pepsin activity,, the protein rapidly re~olds and/or aggregates, which renders it again re~ractory to the proteolytic action o~ pepsin.
The applicant has discovered that i~ proteins are ~irst denatured in 2 2M guanidine thiocyanate at 0~C and the concentration o~ thiocyanate then reduced to 5 2M while at the same time the guanidine ion is maintained 2 2M (by diluting the guanidine thiocyanate into guanidine hydrochloride), the denatured protein r~m~;n¢ in solution, remains denatured, and the enzyme pepsin is e~iciently proteolytically active against the denatured protein in this solution at O'C. The stability o~
pepsin-agarose to this digestion bu~er is such that no detectible degradation in the per~ormance o~ the pepsin column employed by the applicant has occurred a~ter being used to proteolyze more than 500 samples over 1~ years. No pepsin autodigestion takes place under these conditions.
Denaturation without concomitant reduction o~ the binding protein may be accomplished by contacting it (at 0-5~C) with a solution containing 2 molar guanidine thiocyanate pH 2.7, ~ollowed by the addition o~ an equal volume o~ 4 molar guanidine hydrochloride pH 2.7.
Denaturation with disul~ide reduction may be accomplished by contacting the binding protein with a solution containing 2 molar guanidine thiocyanate, 0.7 molar T~EP, 5-20~ H20 (by volume), with the balance o~ volume being acetonitrile, dimethyl sul~oxide, or other water miscible nonaqueous solvent in which the denaturant (e.g. guanidine thiocyanate) and disul~ide bond disrupting agent (e.g., TCEP), i~ used, remain soluble at substantially these concentrations, and such that the solvent system does not ~reeze at the "slow exchange" temperature. The - pH o~ the solution is pre~erably in the range o~ 4.8 - 5.2, optimally 5Ø A~ter this incubation, 2 volumes o~ a 2.5 molar W O 97/41436 - PCT/U'~ C011 guanidine hydrochloride solution is added, with the pH and bu~ering capacity o~ the solution such as to achieve a p~ o~ 2.7 in the ~inal mixture.
Denatured (with or without reduction) binding protein is then passed over a column composed o~ insoluble (solid state) pepsin, whereby during the course o~ the passage o~ such denatured or denatured and reduced binding protein through the column, it is substantially completely ~ragmented by the pepsin to peptides o~ size range 1-2Q amino acids at O C and at pH 2.7.
The e~luent ~rom this column (cont~;n;ny proteolytically-generated ~ragments o~ binding protein) is directly and immediately applied to the chromatographic procedure employed to separate and isolate protein ~ragments, pre~erably analytical reverse-phase HPLC chromatography.
It should be noted that denaturants, besides rendering the binding protein more susceptible to proteolysis, also help dissociate it ~rom its partner.

6. Generation of~ Trlti7lm-Labeled Peptide Fraqments.
To ultimately localize the protein's amides which are ~unctionally labeled with tritium, small peptides bearing the retained tritium label (pre~erably, 5-25 amino acids in size) must be proteol~tically generated ~rom labeled protein and separated ~rom the many other unlabeled peptides generated by ~ragmentation o~ the protein, all under conditions which m'n;m;ze o~-exchange o~ amide tritium ~rom the peptide. Small peptides have little secondary structure and therefore their amides are ~ree to exchange with solvent hydrogen. I~ tritium label is to remain in place on such peptides, proteolysis and puri~ication (e.g., RP-HP~C) conditions must be adjusted to slow such o~-exchange.
The labeled and dissociated binding protein is there~ore~ragmented under slow H-exchange conditions, e.g., by proteolysis with high concentrations o~ a protease which is stable and active with the a~orementioned conditions (e.g., pH 2.7, O~C). ~uitable acid tolerant proteases include pepsin (19), cathepsin-D (37) - Asperqillus proteases (37a-37c), thermolysin (38) and mixtures o~ these proteases. In a pre~erred embodiment, pepsin is used, pre~erably at a concentration of~ 10 mg/ml pepsin at 0~C pH 2.7 ~or 5-30 minutes, pre~erably 10 minutes.
Other physical and chemical ~ragmentation methods may be used provided they are (1)- are compatible with slow H-exchange conditions, (2) do not cause shiEts in the positions o~ the amide labels, and (3) produce a reasonable number o~ ~ragments ~rom the protein o~ interest.
Pre~erably, prior to ~ragmentation o~ the binding protein, binding partner (i~ susceptible to the ~ragmenting agent) is removed, so as not to complicate puri~ication with binding partner ~ragments.
6A. Puri~ication o~ Fragments As acid proteases in general have very broad cleavage speci~icity, they ~ragment the protein into a very large number o~ di~erent peptides. In most protein-binding partner systems studied by tritium exchange, it is likely that the interacting binding sur~aces will contain roughly 10-20 tritium labeled peptide amide which upon proteolysis will result in approximately 1-5 label-bearing peptides, the precise number depending on the inherent ~ragmentation mode o~ the protein under study with the proteases utilized. The number o~ "background," non-labeled peptides (derived ~rom regions o~ the protei~ and binding partner that do not participate ln the binding interaction) generated by the ~ragmentation procedure will be a direct ~unction o~ the s ze o~ the protein. Background peptides will be present in the proteolytic digest in numbers 10-1,000 times greater than will be ~unctionally labeled peptides when proteins with sizes in the range o~ 30-200 kD are proteolyzed.
This large number o~ background peptides causes two di~iculties: First, they must all be cleanly separated ~rom the ~unctionally labeled peptides to allow identi~ication o~ the label-bearing peptides Second, background peptides contain small amounts o~ tritium label and even though the amount o~
label per background peptide is generally less than 1~ o~ that o~ ~unctionally labeled peptides, background peptides are present in much greater amounts and are likely to obscure the presence - o~ ~unctionally labeled peptides and analytical separation.
Given these considerations, only proteins less than 30 k3 W O97/41436 PCT~US96/060Il 33 in size have been success~ully characterized in the past by medium resolution tritium P~ch~nge. Upon acid proteolysis of larger proteins, so many di~erent ~ragments would be bbtained that individual ~ractions ~obtained on a single HPLC separation per~ormed at pH 2.7 would be unacceptably cont~m; n~ ted with background peptides.
Any method of puri~ying the ~ragments which is capable of resolving the mixture while maint~1n;ng slow H exchange condition is acceptable The preferred method is high pressure liquid chromatography (HPLC), especially in reverse phase (RP). (An alternative method is that o~ mass spectroscopy.) The art has overstated the sensitivity o~ the tritium label to pH. Englander (10) reported that at 0~C., the tritium label was most stable ~when the tritiated protein was placed in an untritiated aqueous bu~er) at pH 2.7, and that the rate o~ o~-exchanged increased rapidly (10 ~old per pH unit) as one moved away ~rom that pH. Surprisingly, Applicant ~ound that at 0~C., the label was su~iciently stable to permit analysis even at a pH o~ 2.1. While the acceptable pH range will vary with temperature, and the choice o~ solvent (the optimal pH increases i~ a polar nonaqueous solvent is introduced), the ~act r~m~;nR
that pH was previously considered to be essentially ~ixed. Since the tritium label is stable over a broader pH range, such as 2.1-3.5, it is possible to depart ~rom Englander's recommended pH o~
2.7 in seeking HPLC conditions which result in ef~ective separation o~ the peptide ~ragments.
When the binding molecules are large, so many di~erent ~ragments are obtained after proleolytic digest that some o~ the individual peaks on a single HPLC separation, even at optimized ph, may be heterogeneous.
RP-XPLC resolution o~ co-migrating multiple peptides may be greatly improved by resorting to a two-~;m~n~ional RP-HPLC
separation in which two sequential RP-HPLC separations are per~ormed at substantially di~ferent pH's, e.g. 2.7 and 2.1.
35A two-~;m~n~ional HPLC separation allows high e~iciency puri~ication o~ tritium label bearing-peptides from the enormous - number o~ unlabeled peptides generated by peptic ~ragmentation o~ large proteins. Two-~;m~n~ional separation o~ molecules is W O 97/41436 PCT/U'~ 011 known in the chromatographic art. However, despite ~requent complain~s in the Tritium ~ch~nge literature about resolution problems, 2D separations have not been employed previously in connection with Tritium exchange.
In a pre~erred embodiment o~ the invention, tritium-labeled protein ~ragments are ~irst separated by means capable o~
su~icie~tly resolving the ~ragments, such as by RP-HPLC
(utilizing any o~ a number o~ potential chromatographic support including C4, C18, phenol and ion exchange, pre~erably C18).
10 This separation may be per~ormed at pH 2.1-3.5 and at 4-0~C, more pre~erably, at pH 2.7 and 0~C, which may accomplished by employment o~ any bu~er systems which operate at this pH, including citrate, chloride, acetate, more pre~erably phosphate.
Peptides are eluted ~rom the reverse phase column with a similarly bu~fered gradient of polar co-solvents including methanol, dioxane, propanol, more pre~erably acetonitrile.
Eluted peptides are detected by on-line ultraviolet light absorption spectroscopy per~ormed at ~requencies between 200 and 300 nM, pre~erably 214 nM. Tritium label is detected by scintillation counting o~ a sampled ~raction o~ the HPLC column a~luent. Peptides bearing label that has been speci~ically protected ~rom o~f-exchange by complex ~ormation with binding partner are identi~ied by comparing the speci~ic activity o~ each labeled peptide to the speci~ic activity of the same peptide prepared ~rom protein sub~ected to identical on/o~ exchange, proteolysis and HPLC conditions, but which have been o~-exchanged withou~ added binding partner.
HPLC fractions containing peptides with such ~unctionally labeled amides are then subjected to a second ~1~ m~n~:ion RP-MPLC
30 separation which may be per~ormed at pH 2.1-3 5 and 4-O~C, more pre~erably, at pH 2 1 and 0~C, accompanied by any bu~er systems which operates at this pH, including citrate, chloride, acetate, phosphate, more pre~erably TFA (0.1-0.115~). Peptides are eluted ~rom their reverse phase column with a similarly bu~ered gradient o~ polar co-solvents including methanol, dioxane, propanol, more pre~erably acetonitrile. Eluted peptides are - detected, tritium measured and ~unctionally labeled peptides identi~ied as in the ~irst HPLC ~;men~ion described above.

W O97/41436 - PCT~US96/06011 Functionally labeled peptides are isolated (collection of the appropriate ~raction o~ column e~luent), water, acetonitrile, and TFA removed by evaporation, and the remaining puri~ied peptides each characterized as to its primary amino acid structure by conventional technigues, e.g., amino acid analysis o~ complete acid hydrolysates or gas-phase Edman degradation microsequencing. Re~erence is then made to the previously known amino acid seguence o~ the intact protein to in~er the location o~ the tritium-labeled peptides within the intact protein's primary sequence. Employment o~ TFA bu~er in the second ~imPn~ion has the additional advantage that no residual salt (i.e. phosphate) remains a~ter solvent evaporation. Residual phosphate ~requently inter~eres with the chemical reactions required ~or amino acid analysis and Edman degradation, a problem obviated by the use o~ volatile TFA in the second ~nqion bu~er.
Most pre~erably, proteolytic digests are ~irst separated at pH 2.7 in phosphate bu~ered solvents and each eluted peptide peak ~raction which contains tritium-labeled amides is identi~ied, collected, and then subjected to a second HPLC
separation per~ormed in tri~luoracidic acid (TFA)-bu~fered solvents at pH 2.1.

7. Hiqh Resolution Sublocalization of Labeled Amides Wi thin Label -Bearinq Peptides .
25To routinely localize peptide amide tritium label to the single amino acid level, applicant systematically cleaves every peptide bond within a puri~ied label-bearing peptide. Slow H-~rh~nge conditions must be used ~or this proteolysis as the small peptides generated have no stable con~ormational structure and rapid loss o~ tritium label ~rom the amides would occur i~
rates o~ exchange were not slowed, e.g., by ambient acidic pH.
Most known acid-reactive proteases cleave peptides in a - basically nonspeci~ic manner similar to that of pepsin; studies employing other pepsin-like proteases have not proved to be o~
signi~icant utility in increasing resolution o~ labeled amides.
- ~ special class o~ acid-reactive proteases, the carboxypeptidases, are able to generate all reguired sub~ragments CA 02228404 l998-0l-30 W O 97/41436 PCTrUS96/06011 o~ pepsin-generated peptides in quantities su~icient ~or high resolution tritium localization. Many carboxypeptidases -are active at pH 2.7 and sequentially cleave amino acids ~rom the carboxy terminus of peptides. Such enzymes include carboxypeptidase P, Y, W, and C (39). While carboxypeptidases have been used ~or limited carboxy-term;n~l sequencing of peptides, o~ten at pH in the range of 2.7 (40), their use in tritium exchange techniques has not been disclosed. The need to minimize tritium losses ~orbids the use o~ carboxypeptidases which are inactive in acidic (pH 2.7) bu~fers, such as carboxypeptidases A and B. However, carboxypeptidase-P, Y, and several other acid-reactive carboxypeptidases (W,C) are suitable ~or proteolysis of peptides under acidic conditions (39). The tritium exchange art has ~ailed to recognize the utility of carboxypeptidases to tritium exchange studies, possibly because the carboxypeptidases are even more nonspeci~ic in the types of peptide bonds they cleave than are pepsin-like proteases and there~ore might have been thought to result in inadequate recovery o~ any single sub~ragment.
Furthermore, chemical procedures employing penta~luoropropionic anhydride can produce sets o~ C-t~rm; n~ 1 -truncated peptide ~ragments under slowed amide ~h~nge conditions (see below, 41,42).
In the preferred embodiment, tritium-exchange-labeled proteins are nonspecifically fragmented with pepsin or pepsin-like proteases, the resulting tritium-labeled peptides isolated by two-~m~n~ional HPLC and these in turn exhaustively subfragmented by controlled, step-wise diqestion with acid-(i.e., enzymatically active under acidic conditions) exopeptidases and/or by chemical means (see below). These digests are then analyzed on RP-HPLC per~ormed at 0~C in TFA-containing bu~ers (pH 2.1) and each o~ the generated sub~ragments (typically 5-20~
is then identi~ied. The identity of each of the several subfragments maybe determined by any suitable amino acid analysis, peptide sequencing, or through the use of synthetic HPLC mobility marker peptides, and the amount o~ tritium label - attached to each sub~ragment truncated peptide determined by scintillation counting. In this manner, the precise location, W O 97/41436 - PCT~US96/~6011 within the protein, o~ each peptide amide that is ~unctionally labeled with tritium by virtue o~ its interaction with binding partner is det~rm;n~d By consideration of the tritium content o~ each o~ the identi~ied sub~ragments the amide hydrogens which had been replaced by tritium during the "in-exchange" step may be in~erred. It should be noted that the purpose o~ the carboxypeptidase treatment is to generate the sub~ragments; the method does not require use o~ carboxypeptidase to sequence the ~ragments or sub~ragments. Pre~erably, the sequence o~ the binding protein, or at least o~ the material portion thereo~, is known prior to commencement o~ the present method. However, it may be determined at any time, even a~ter the sub~ragmentation, although the data gleared ~rom the subfragmentations cannot be properly interpreted until the sequences o~ a least the source is known.
Controlledsequential carboxy-termin~l digestion o~ tritium-labeled peptides with carboxypeptidases can be per~ormed under conditions which result in the production o~ analytically su~icient quantities o~ a set o~ carboxy-terminal truncated daughter peptides each shorter than the preceding one by a single carboxy-te~m;n~l amino acid. As each carboxy-terminal amino acid o~ the ~unctionally labeled peptide is sequentially cleaved by the carboxypeptidase, the nitrogen which ~ormed the slow-exchanging peptide amide in the intact peptide bond is converted to a rapidly exchanging secondary amine, and any tritium label at that nitrogen is lost ~rom the peptide within seconds, even at acidic pH. A di~erence in the molar quantity o~ tritium label associated with any two sequential subpeptides implies that label is localized at the peptide bond amide which di~ers between the two subpeptides.
In a pre~erred embodiment, synthetic peptides are produced (by standard peptide synthesis techniques) that are identical in primary amino acid sequence to each o~ the ~unctionally labeled pepsin-generated peptides identi~ied in Step 6. The synthetic peptides may then be used in prel;m;n~y carboxypeptidase digestion (pH 2.7, 0~C) and XPLC (in TFA-bu~ered solvents) - studies to determine; 1) the optimal conditions o~ digestion time and protease concentration which result in the production and W O 97141436 PCT/U'~ 011 identi~ication digestion on all possible carboxypeptidase ~roducts o~ the peptide under study; and 2) the HPLC elution position (mobility) o~ each carboxypeptidase-generated sub~ragment o~ synthetic peptide.
5To ~acilitate this latter procedure, a set o~ re~erence peptides may be produced consisting o~ all possible carboxy-terminal truncated daughter peptides which an acid carboxypeptidase could produce upon digestion o~ a "parent"
peptide. These serve as HPLC mobility identity st~n~rds and allow the deduction o~ the identity o~ daughter peptides actually generated by carboxypeptidase digestion. Certain daughter peptides may be enzymatically produced in quantities insu~icient ~or direct amino acid analysis or sequencing, but their HPLC
mobility can be measured and compared to that o~ the synthetic peptides. Peptides can be detected and quanti~ied by standard in-line spectrophotometers (typically W absorbance at 200-214 nM) at levels well below the amounts needed ~or amino acid analysis or gas-phase Edman sequencing.
A~ter these prel;m~n~ry studies, the pepsin-generated HPLC
isolated, ~unctionally labeled peptide (prepared in Step 6) is then carboxypeptidase digested and analyzed under the ~oregoing experimentally optimized conditions, the identity o~ each ~ragment detPrmlned (by peptide sequencing or by re~erence to the mobility o~ re~erence peptide mobility marker) and the amount o~
tritium associated wi~h each peptide sub~ragment determined.
Alternatively, a chemical technique may be used ~or the successive carboxy term; n~ 1 degradation o~ peptides under slowed tritium exchange conditions. Tritium-labeled peptides in HPLC
bu~ers are held at -35~C and solvents removed ~y cryosublimation 30(40a, 40b; vacuum at 1-20 millitorr, solvents collected in a liquid nitrogen trap). The dried peptide is then reacted with vapor phase penta~luoropropionic acid anhydride (PFPA) as described in (54,55) except that the peptide temperature is kept at -35~C ~or times up to 3 hours. PFPA is then removed by vacuum 35and the ~ragmented peptide made to 50 mM PO4 pH2.7, and analyzed by HPLC.
-In general, the known aminopeptidases are not able to sequentially degrade a peptide under slow hydrogen exchange W O 97/41436 PCT~US96/06011 conditions. However, i~ an acid-reactive aminopeptidase is discovered in nature, or produced by mutation o~ a known aminopeptidase, there is no reason that an aminopeptidase-can not be used in place o~ the presently pre~erred carboxypeptidase.
In that event, the stepwise degradation will begin at the N-t~rm;n~l, rather than the c-t~rm;n~l, o~ each analyzed peptide ~ragment.
It should be noted that by using polar, nonaqueous high concentrations o~ cosolvents to shi~t the pH~ o~ the H-exchange rate, a greater variety o~ reagents may be used than would otherwise be the case. A cosolvent o~ particular interest in this regard is glycerol (or other polyols), as it is unlikely to denature the enzyme when employed at the high concentration to substantially shift the p~ min.

15 8. Optimization of on and o~ exchanqe times.
Each peptide amide hydrogen associated with the protein-binding partner interaction sur~ace has a unique exchange rate with solvent tritium in the native ~olded, unliganded state, which is then shi~ted to another distinct exchange rate once protein-binding partner complex ~ormation has occurred. The signal to noise ratio (ratio of tritium ~unctionally bound to this peptide amide over total background tritium bound to all other peptide amides in the protein) can be optimized by a knowledge o~ the exchange rates o~ this amide hydrogen in the native unliganded protein and in the protein-binding partner complex.
An amide hydrogen with an exchange hal~ e o~ one minute in the protein's native, unliganded state and 10 minutes in the liganded state might be optimally studied by on-exchanging the receptor protein ~or 2 minutes (2 hal~-lives o~ on-exchange time will result in incorporation o~ tritium at 75~ o~ the maximal possible equilibrium labeling o~ the peptide amide) ~ollowed by - 10 minutes o~ o~-exchange in the liganded state (50~ o~ on-exchanged label will remain on the ~unctionally labeled peptide amide and less than 0.1~ o~ on-exchanged label will remain on - each o~ the background labeled peptide amides).
-To measure the exchange rates o~ a parti~ular ~unctionally CA 02228404 l998-0l-30 W O 97141436 PCT/U~ 6011 lahelable peptide amide as i~ exists in the native, unliganded protein, aliquots o~ protein are on-~ch~nged ~or varying times (0.5 seconds to 24 hours), bound to binding partner, and then o~-exchanged ~or a ~ixed time, pre~erably 24 hours. A~ter pH
2.7, O~C proteolytic digestion and HPLC separation, radioactivity associated with the peptide ~ragment containing the peptide amide under study is measured. The amount o~ the radioactivity which represents background (amides which are not ~unctionally labeled) is determined by measuring the amount of label associated with the same peptide when the protein is on-exchanged ~or the same duration but oi~-exchanged Eor 24 hours in the absence oE added ligand prior to proteolysis and HP~C analysis. Speci~ic radioactivity associated with the amide is determined as a function o~ on-exchange time, and the hal~ e o~ (on) exchange o:E the amide in the unliqanded protein calculated.
To determine the ~hAnge rate o~ the same peptide amide when it is in the protein-binding partne~ complex, protein is on-exchanged for a fixed, long period o~ time (preferably 24 hrs) complexed with binding partner, o~-exchanged ~or varying times (pre~erably 10 seconds to 4 days), acid proteolysed, and HPLC
analyzed as above. Specific radioactivity associated with the amide is det~rm~n~d as a ~unction o~ o~-exchange time, and the hal~ e o~ (o~)-exchange o~ the amide in the liganded protein calculated. With this in~ormation the times o~ on and o~-exchange are adjusted to optimize the signal/noise ratio for eachof the amides ~unctionally labeled in the protein-binding partner system under study.

9. Modelinq of Receptor Liqand Contact Surfaces.
Studies identical in design to those described above (1-8) may also be per~ormed on the corresponding binding partner protein (the binding partner protein is on-exchanged, liganded to receptor protein, o~-exchanged, etc.), resulting in the identi~ication o~ the amides o~ the binding partner which are slowed in exchange by virtue o~ interaction with receptor protein. The knowledge o~ the identity o~ the precise contact ~ peptides in both protein and binding partner may be used to produce computer-assisted models ~or the complementary 3-W O 97/41436 PCT/U'~ C011 ~;m~nsional structures o~ the protein and binding partner sur~aces.
Construction o~ these models is aided by additional in~ormation provided by ~ the invention which allows the identi~ication o~ a subset o~ peptide amides on the protein's binding sur~ace which are likely to ~orm hydroqen bonds with acceptor residues on the cognate binding protein contact sur~ace.
While most o~ the peptide amides present on the native, uncomplexed protein or binding partner interaction sur~aces can be expected to be hydrogen bonded to other portions o~ the same protein, a ~raction of these peptide amides, possibly approaching 50~, may be hydrogen bonded only to solvent. As most protein-binding partner contact sur~aces are highly complementary to each other, it is likely that upon complex ~ormation solvent water is removed ~rom the interaction sur~aces, and amides previously hydrogen bonded to water will ~orm new hydrogen bonds to the complementary sur~ace o~ the partner. This subset o~ binding sur~ace amides is readily identi~ied in our studies (Step 8) as they will have an exchange rate in the protein1s native, unliganded state o~ 0.5 seconds at pH 7.0 and 0~C. These amides can ~orm hydrogen bonds with the complementary sur~ace only i~
their hydrogens are oriented in the direction o~ the complementary sur~ace. This in turn places orientation constraints on the entire associated peptide bond and to a lesser degree the side ~h~ ~ n~ o~ the two ~lanking amino acid residues o~ each such amide. Application o~ these constraints to the ~oregoing models o~ interaction sur~ace structure allow higher resolution modeling o~ the 3-~mPn~ional structure of the protein-binding partner ligand interaction sur~aces.

10. Automation o~ the procedures required for the performance of enzymatic deqradation and HPLC analysis under slowed tritium exchanqe conditions.
- While digestion and analysis procedures are per~ormed at ~~C, analytical samples o~ tritium exchange-labeled peptides must be stored at temperatures o~ approximately -60 to -80~C i~
- unacceptable losses o~ label ~rom the peptide are to be avoided over intervals o~ hours to weeks. Tritium exchange continues in WO 97/41436 PCT/U~5'.'0Il ~rozen samples in a m~nn~r inversely related to temperature but ef~ectively stops at temperatures o~ approximately -70~C. At present, tritium exchange analysis is per~ormed by ~n~l~71y removing samples ~rom -70~C storage, melting them m~nl~lly at 0~C, m~nll~l addition o~ reagents ~bu~ers, enzymes) and m~n~
injection of samples onto the HPLC column. These manipulations are labor intensive and expose the samples to inadvertent heating during handling. I~ HPLC-separated peptides are to be collected and stored ~or ~uture study, they are manually collected and stored at -70~C. No presently available robotic HPLC autosampler has the capability o~ performing the necessary manipulations on samples stored in the ~rozen state.
A Spectraphysics AS3000~ autosampler may be modi~ied so as to allow automation o~ these steps. These pre~erred modi~ications were: inclusion o~ a solid dry ice bath ln which samples are stored until analysis; use o~ modi~ied ~luidic syringes which operate reliably at 0~C; control o~ the autosampler by an external computer; and placement o~ the autosampler HPLC column and spectrophotometer within a 0~C
re~rigerator. Under computerized control, the autosampler's mechanical arm li~ts the desired sample ~rom the -70~C bath, and places it in a heater/mixer which rapidly melts the sample at 0~C. The liqui~ied sample is then automatically injected onto the HPLC column. Operation o~ HPLC pumps, on-line radiation counter and data acquisition is similarly automated.
To collect tritium-labeled, HP~C-separated peptides under slowed exchange conditions, a Gilson-303~ ~raction collector (also present in the 0~C re~rigerator) has been modi~ied so that the sample collection tubes are immersed in a dry ice bath.
Computer-directed diversion o~ desired HPLC e~luent ~ractions into these prechilled tubes results in rapid ~reezing o~ the desired tritium-labeled peptides to -70~C.
Deu teri um Exchanqe Embodimen ts:
In another em~odiment, functionally labeled proteolytic ~ragments, generated ~rom a protein that has been ~unctionally labeled with deuterium (rather than tritium) prior to recep~or-- ligand complex ~ormation, are analyzed by mass spectroscopy, conducted under conditions which m;n;m;ze o~-exchange o~ peptide W 097/41436 PCT/U~G~'a~0~1 43 amide deuterium ~rom peptide ~ragments and allow the direct determ; n~ tion o~ the location of ~unctionally attached label within a peptide in the size range 3-30 amino acids.
Mass spectroscopy has~become a standard technology by which the amino acid sequence of proteolytically generated peptides can be rapidly determined (43). It is commonly used to study peptides which contain amino acids which have been deuterated at carbon-hydrogen positions, and thereby determine the precise location o~ the deuterated amino acid within the peptide's primary sequence. This is possible because mass spectroscopic techniques can detect the slight increase in a particular amino acid's molecular weight due to the heavier mass o~ deuterium.
McCloskey, et al (44) discloses use of deuterium exchange o~
proteins to study con~ormational changes by mass spectrometry.
The applicant has devised a deuterium-exchange technique essentially identical, in steps 1-5, to the tritium exhange technique described above except that on-exchange is per~ormed in deuterated water (preferably 80-99~ mole ~raction deuterated water). This modi~ied procedure, a~ter addition o~ binding partner and o~-exchange, speci~ically labels with exchanged deuterium the peptide amides which make up the interaction surface between protein and binding partner. Proteolytically generated ~ragments o~ protein ~unctionally labeled with deuterium are identified, isolated, and then subjected to mass spectroscopy under conditions in which the deuterium remains in place on the ~unctionally labeled peptide amides. Standard peptide sequence analysis mass spectroscopy can be per~ormed under conditions which m;n;m;ze peptide amide proton exchange:
samples can be maintained at 4~C to zero degrees C with the use 3 0 of a re~rigerated sample introduction probe; samples can be introduced in buffers which range in pH between 1 and 3; and analyses are completed in a matter o~ minutes. MS ions may be made by MALDI (matrix-assisted laser desorption ionization) - electrospray, ~ast atom bombardment (FAB), etc. The carboxypeptidase may act before or simultaneously with the ionization events. Subfragments are separated by mass by, e.g., - magnetic sector, quadropole, ion cyclotron, or time-o~-~light methods. For MS methods generally, see Siuzdak, G., Mass CA 02228404 l998-0l-30 W O 97/41436 PCT/U~

Spectrometry for Biotechnology (Academic Press 1996).
Since deuterium is not radioactive, the deuterium-labeled peptides must be identified by other means, such as mass spectrometry (their molecular weight will be greater than that o~ predicted for the same peptide without such a label).
If desired, the same binding protein: binding partner complex may be studied both by tritium exchange (which need only be to medium resolution) and by deu~erium exchange. The tritium exchange method will identify the relevant fragments. Since the HPLC mobilities o~ these tritium-labeled ~ragments will then be known, the corresponding deuterium-labeled ~ragments can be identified by their common mobilities and then sub~ragmented, etc.
In a preferred embodiment, separate tritium and deuterium exchange runs are avoided. Instead, the deuterated water is supplemented with tritiated water, e.g. the solvent is 98~ mole ~raction deuterated water and 2~ mole fraction tritiated water (e.g., 50 Ci/ml) As a result, the fragments are labeled both with deuterium and tritium, and the relevant fragments identified 2~ by their tritium-imparted radioactivity. The subfragments are still analyzed by mass spectroscopy for the presence o~
deuterated label (with appropriate correction for the relatively small amount of tritium also present). The purpose of the tritium is to radioactively tag peptide ~ragments containing binding sur~ace residues. However, the exact residues involved are identified by MS analysis of deuterium bearing peptides that have been ~urther digested with acid-reactive carboxypeptidases, allowing identification of the deuterated residues of the radioactive peptides.
In a preferred embodiment, receptor-binding partner complexes functionally labeled with deuterium and tritium at their interaction surface are (under slowed exchanged conditions as described above for high resolution tritium exchange analysis) pepsin digested, subjected to rpHPLC in 0.1~ TFA cont~;n;ng buffers and column effluent cont~;n;ng tritium labeled peptides subjected to mass spectroscopic analysis. To more precisely - localize the deuterium label within each peptide, mass spec~rometry is performed on labeled-proteolytic ~ragments, that , W O 97/41436 - PCT/U~ 011 ~5 are progressively ~urther digested (under slowed exchange conditions) with acid-reactive carboxypeptidases (41). This digestion can be per~ormed be~ore introduction o~ the sample into the mass spectrometer, or continuously in situ while the sample is held in the mass spectrometer. As digestion proceeds, molecular ions o~ each of the resulting enzyme-generated carboxy-terminal truncated peptide sub~ragments is detected by the mass spectrometer, and its molecular weight compared to that known ~or the undeuterated ~orm o~ the same peptide ~ragment. Peptide ~ragments which bear ~unctionally attached deuterium are identi~ied by an increase in their molecular weight o~ one atomic unit when compared to the same peptide ~ragment generated from undeuterated receptor-binding partner. Su~icient sub~ragmentation and analysis as above results in the deduction o~ the protease-generated ~ragments that have ~unctionally-bound deuterium. Thereby, the location o~ each deuterated amide within the peptide is determined.
In vivo Analysis.
In situ analysis o~ protein-binding partner interactions is possible in vivo. The protein, while present in its native environment as a component o~ an intact living cell, or as a component o~ a cellular secretion such as blood plasma, is on-exchanged by inc~bating cells or plasma in physiologic bu~ers supplemented with tritiated (or deuterated) water. The binding partner is then added, allowed to complex to the cell or plasma-associated protein, and then o~-exchange initiated by returning the cell or plasma to physiologic conditions ~ree o~ tritiated (or deuterated) water. During the o~-exchange period (hours to days) the formed protein-binding partner complex is isolated ~rom the cell or plasma by any puri~ication procedure which allows the complex to remain continuously intact. At the end o~ the appropriate o~-exchange period, ~ragmentation and analysis o~
puri~ied complex proceeds as above.
This analytic method is especially appropriate ~or proteins which lose substantial activity as a result o~ puri~ication, as the binding site is labeled prior to puri~ication.
- Bindinq ~ite Analysis by Indirect Hydroqen Exchange In the methods described above, the entire sur~ace o~ the W O97/41436 PCTrUS96/06011 = 46 protein is labeled initially, and label is then removed ~rom those surfaces which remain solvent exposed after formation of the complex of the binding protein and its binding partner. The binding site o~ the protein is occluded by the binding partner, and label is there~ore retained at this site.
When the complex is formed, the binding protein may undergo changes in conformation (allosteric changes) at other sites, too.
If these changes result in segments o~ the protein being buried which, previously, were on the surface, those segments will likewise retain label.
It is possible to distinguish binding site residues from residues protected ~rom "of~-exchange" by allosteric ef~ects.
In essence, the binding partner, rather than the binding protein, is labeled initially The binding protein is labeled indirectly as a result of trans~er o~ label from the binding partner to the binding protein. Such transfer will occur principally at the binding surface.
This procedure will functionally label receptor protein amides if they are slowed by complex formation and are also in lntimate contac~ with the bindinq partner in the complexed state.
Receptor protein amides that are slowed because of complex formation-induced allosteric changes in regions of the protein which are not near the protein-binding partner interaction surface will not be labeled. This procedure may be performed as ~ollows:
1) binding partner is added to tritiated water (preferably of high specific activity) to initiate trltium exchange labeling o~ the b; n~; ng partner 2) After sufficient labeling is achieved, binding partner is separated from the excess of solvent tritium under conditions which produce m; n;m~l loss of tritium label from the binding partner. This can be accomplished by, e.g., a) shifting the buffer conditions to those o~ slowed ~h~nge (0~C, acidic pH) followed by G-25 spin column separation of the binding partner into tritium-free buffer or b) employing stopped-flow techniques in which the on-exchange mixture is rapidly diluted with large - volumes of tritium free buffer.
3) the tritium-labeled binding partner, now essentially free W O 97/41436 PCTrUS96/06011 o~ excess solvent tritium, is added to receptor protein and conditions adjusted to allow spontaneous reversible (equilibrium) complex formation to take place between the two. The conditions o~ temperature and pH should also allow, and preferably maximize, the speci~ic transfer o~ tritium label from the labelled binding partner to amides on the binding protein's interaction surface with partner. Typically, the pH will be 5-8 (conducive to ligand binding) and the temperature 0-37~C. Initially, use of~ pH 7 and 22~C. is recnmm~n~ed, the transfer being controlled by controlling the incubation time. A typical trial incubation time would be 24 hours. These conditions o~ pH, temperature and incubation time may o~ course be varied.
~ ) The complex is then incubated for periods of time sufficient to allow trans~er o~ tritium label ~rom the labeled binding partner to the receptor protein. During this incubation period, tritium which has on-exchanged to regions of the binding partner that are distant ~rom the receptor-binding partner interaction sur~ace will leave the binding partner by exchange with solvent hydrogen and be rapidly and highly diluted in the large volume o~ solvent water, thereby preventing its ef~icient subsequen~ interaction with the binding protein. However, tritium label that has been attached to binding partner amides present within the (newly ~ormed) protein-binding partner interaction surface will be capable o~ exchanging o~ of the binding partner only during the brief intervals when the interaction surface is exposed to solvent water, i.e., when the complex is temporarily dissociated. When so dissociated and solvent exposed, a portion of tritium present on amides within the binding partner's interaction sur~ace will leave the surface and for a brie~ time, remain within the proximity o~ the surface.
Given the rapid (essentially diffusion limited) rebinding of binding protein and partner, much o~ the released tritium that (briefly~ remains within the environs of the partner's binding - sur~ace will in part exchange with amides on the (~uture) interaction sur~ace of the approaching binding protein molecule that subsequently binds to the binding partner. Once such - binding occurs, the transferred tritium is again protected from exchange with solvent until the complex dissociates again. The W O97/41436 PCT/U',~'~C~ll result will be the progressive trans~er o~ a portion o~ the tritium ~rom the binding partner interaction sur~ace to exchangeable amides on the cognate protein interaction sur~ace.
Amides whose exchange~~ates are conformationally slowed each time complex ~ormation occurs can also become labelled with tritium, but they will do so at a much slower rate than amides within the binding sur~ace, as they are located more distant ~rom the high concentration o~ tritium "released" at the interaction sur~ace with each complex dissociation event. The e~iciency o~
trans~er is roughly inversely proportional to the cube o~ the distance between such con~ormational changes and the binding sur~ace.
The binding protein-tritiated binding partner complex incubation conditions are adjusted to optimize speci~ic interaction sur~ace amide tritium trans~er (SISATT) ~or a particular binding protein-partner pair. SISATT is de~ined as the ratio o~ the amount o~ tritium (CPM) trans~erred ~rom binding partner to binding protein peptide amides previously determined (by the technique o~ Claim 1) to undergo slowing o~ amide hydrogen exchange upon binding-protein partner complex ~ormation divided by the total tritium (CPM) trans~erred ~rom binding partner to all peptide amides in the binding protein.
5) A~ter an incubation period that allows and pre~erably maximizes SISATT, the conditions o~ slow hydrogen exchange are restored, the complex is dissociated and the binding protein ~ragmented. Fragments o~ bindin~ ~rotein (as opposed to the initially labeled binding partner) that bear tritium label are identi~ied, and ~urther characterized as previously described.
Alternatively, deuterium is used instead o~ tritium as the label. Deuterium has the advantage o~ allowing a much higher loading o~ label (since deuterium is much cheaper than tritium) It is possible, also, to directly label the binding partner with deuterium and the b; n~; ng protein with tritium. As a result, both the binding site and allosterically buried amides o~ the binding protein will be tritiated, but only binding site amides will be deuterated.
- The indirect method is especially applicable to study o~
I proteins which undergo substantial con~ormation o~ changes a~ter, W O 97/41436 PCT/U~

or in the course o~ binding, such as insulin and its receptor.

Compositions A~ter det~rm; n; ng the~binding sites o~ a binding protein or a binding partner, by the present methods (alone or in conjunction with other methods), the in~ormation may be exploited in the design o~ new diagnostic or therapeutic agents. Such agents may be ~ragments corresponding essentially to said binding sites (with suitable linkers to hold them in the properspatial relationship if the binding site is discontinuous), or to peptidyl or non-peptidyl analogues thereo~ with the similar or improved binding properties. Or they may be molecules designed to bind to said binding sites, which may, i~ desired, correspond to the paratope o~ the binding partner.
The diagnostic agents may ~urther comprise a suitable label or support. The therapeutic agents may ~uther comprise a carrier that enhances delivery or other improves the therapeutic e~ect.
The agents may present one or more epitopes, which may be the same or di~erent, and which may correspond to epitopes o~
the same or di~erent binding proteins or binding partners.

Examples As a demonstration o~ the practical use o~ this technology, Applicant has studied the interaction o~ human hemoglobin with two di~erent monoclonal antibodies known to be reactive with de~ined and previously identi~ied subregions o~ the hemoglobin binding protein haptoglobin. For these studies, I employed monoclonal antibody ~6-1-23456 (speci~ic ~or the human hemoglobin chain; epitope centered on or about ~6Glu and monoclonal antibody ,B121 (speciEic ~or the human hemoglobin ~ chain in the region o~ residue ~121), both antibodies being the generous gi~t o~ C R. Kie~er, Medical College o~ Georgia, Augusta, Georgia (51). ~llm~n haptoglobin was obtained from Calbiochem - Corporation, La Jolla, Cali~ornia.

Preparation o~ hemoqlobin: Blood was drawn ~rom a normal ~ donor into sodium heparin at 10 U/ml. Red blood cells were washed ~ive times in cold phosphate bu~ered saline (PBS) (pH

CA 02228404 l998-0l-30 W 097/41436 PCT/U',~/C6Cll 7.4) with the bu~y coat aspirated a~ter each wash. An equal volume cold distilled water was added to the washed cell pellet to lyse cells, and then a one-hal~ volume o~ cold toluene was added with vigorous vortexing. This mixture was centri~uged for 30 minutes in a cold Sorvall centri~uge (Dupont) rotor at 15,000 rpm (33,000xg). The hemoglobin (middle) layer was removed and the centri~ugation and hemoglobin decantation repeated. The isolated hemoglobin was dialyzed against ~our changes o~ cold 0.1M sodium phosphate, 0.5~ NaCl pH 7.4. A~ter dialysis, the sample was treated with carbon mono~;de ~or 15 minutes. Final hemoglobin concentration was measured by using a molar extinction ~or heme at 540nm o~ 14,270. The preparation was stored ~rozen in aliquots at -70~C.

Preparation of pepsin: Porcine pepsin (Worthington Biochemical Corp.) was dissolved at 10 mg/ml in 50 mM sodium acetate pH 4.5 and dialyzed against the same solution to remove proteolytic ~ragments. It was stored ~rozen in aliquots at -70~C

Tritium exchanqe: All steps were per~ormed at 0~C. On-~x~h~nge was initiated by mixing equal volumes (5 ~l) o~ isolatedhemoglobin (300 mg/ml) and tritiated water (50 Ci/ml) and the mixture incubated ~or ~our hours. Ali~uots o~ this mixture (1.3 ~1) were then added to equimolar quantities o~ either monoclonal ~1~6, monoclonal ,G~121, haptoglobin, (all at 10 mg/ml in PBS, pH 7.4, in a ~inal incubation volume o~ 75 ~l) or added to 75 ~l o~ PBS
alone. These hemoglobin-ligand mixtures were then immediately applied to 2 ml Sephadex~ G-25 spin columns and centri~uged 4 minutes at 1100xg. Spin columns were prepared by ~illing 3 ml polypropylene columns (~isher Scienti~ic) with 2 ml o~ Sephadex G-25 ~ine equilibrated in PBS pH 7.4 plus 0.1~ Triton* X-100.
Columns were pre-spun at 110Oxg ~or 2 minutes just be~ore use.
A~ter column separation, samples were o~-exchanged by incubation ~or a period o~ 40 hours, ten times the length o~ on-exchange.
Samples were then hydrolyzed with pepsin. Typically, 25 ~l of o~-exchanged mixture cont~;n;ng 70 ~g o~ hemoglobin was added to 10~g pepsin in 110~1 o~ 0.lM NaPO4 pH 2.7 plus 2.5~1 0.5M

W O 97/41436 PCTrUS96/06011 H3P04, the mixture incubated on ice ~or 10 minutes and then injected onto the HP~C column. An aliquot of on-exchanged hemoglobin was immediately adjusted to pH 2.7, passed over a pH
2.7 (0.1 M NaP04 pH 2.7) also proteolyzed and analyzed as above without a period of off-exchange. To measure on-exchange rates of specifically labeled amide protons, hemoglobin was on-exchanged as above but with time intervals ranging from 10 sec -18 hours, reacted with ligand, and off-exchanged for 18 hours.
Samples were then proteolyzed, subjected to HPLC as below, and specific label on peptides quantified as a function of on-exchange time.

Hiqh pressure liquid chromatoqraphy: Digested samples were analyzed on a Waters HPLC unit modi~ied by putting the column and injector under melting ice. Mobile phase was prepared using Barnstead nanopure water, Aldrich ultrapure sodium phosphate, J.T. Baker ultrex grade HC~ and HPLC grade acetonitrile from Burdick & Jackson. Mobile phase consisted of 50 mM NaPO4 pH 2.7 (solvent A) and a mixture o~ 20~ 50 mM NaP~4 and 80~ acetonitrile (ACN) final pH 2.7 (solvent B). Separation of peptides was achieved using a 30 cm Phenomenex Bondclone 10 C18 column. The gradient program started at 100~ A 0~ B and altered the client to 83~A, 17~B over 3.4 minutes. From 3.4 to 6.7 minutes the system ran at a constant 83~A, 17~B and from 6.7 to 73.3 minutes the program implemented a linear increase in ~B ~rom 17~ to 51~.
Absorbance was monitored at 214nm with a Waters model 441 detector.
For second ~;m~n~ion separation, peptide peaks bearing speci~ic label isolated as were collected at 0~C, stored ~rozen at -70~C, thawed at 0~C, mixed with an equal volume of lOOrnM PO4 pH 2.7, and subjected to HPLC as above, except that ~u~er A was 0.115~ tri~luoracetic acid (TFA) in H20 and bu~fer B was 80~ ACN, 20~ H20, 0.1~ TFA. Peaks bearing speci~ic radiolabel were identified and isolated.

Sample collection: HPLC e~luent was collected at the HPLC
detector outflow with a Gilson model 203* fraction collector.
Samples (100 to 400 ~ractions per run) were collected and W O 97/41436 - PCT~US96/06011 radioactivity measured by adding ~ive volumes o~ Aquamix (ICN
Radiochemicals) ~ollowed by scintillation counting. In other studies, on-line liquid scintillation counting was per~ormed using a B-RAM ~low radiation detector (INUS Inc.).

Pe~tide iden~ification: HPLC-isolated peptide were analyzed by both gas phase Edman sequencing and amino acid analysis at the UCSD protein sequencing ~acility.

RESULTS
~emoqlobin-monoclonal antibody epitope ma~ping. Hemoglobin was on-exchanged ~or ~ hours and then either proteolyzed without a period o~ o~ exchange (Fig. 1, panel B), mixed with equimolar quantity O~ ~6 monoclonal and then o~f-exchanged ~or 40 hours (Fig. 1 panel C), mixed with monoclonal ~121 and o~-exchanged ~or 40 hours (data not shown) or o~-exchanged 40 hours in the absence o~ added antibody (Fig. 1, panel D). When labeled hemoglobin is ~m~ned without a period o~ o~ exchange (Fig. 1, panel B), at least 17 radiolabeled peaks were resolved, which generally corresponded to the peaks seen in the optical density trace o~ the same HPLC run (Fig. 1, panel A). When labeled hemoglobin was allowed to ~ully o~ exchange without the presence o~ a protecting monoclonal antibody, all radiolabeled peaks disappeared (Fig. 1, panel D). However, when labeled hemoglobin was o~f-exchanged in the presence of the ~6 monoclonal, a single uni~ue peak bearing radiolabel was seen indicating that this ~raction contains the ~6 monoclonal antigenic epitope (Fig. 1, panel C).
When this peak was subjected to second ~;m~n~ion HPLC in TFA-containing solvents under slowed proton exchange conditions, two peptides were resolved by optical density at 214 nM, with only one of these bearing radiolabel (see Figure 2). This label-bearing peptide was ~ound by gas phase microse~uencing and amino acid analysis to represent residues 1-14 o~ the hemoglobin beta chain. Measurement o~ on-exchange rates o~ labeled amides in this peptide demonstrated two rate classes, both o~ equal size;
one which ~h~nged on with a hal~ e o~ less than 10 seconds, and another with a hal~ e of approximately 1 hour. Speci~ic CA 02228404 l998-0l-30 W O 97/41436 PCTrUS96/06011 activity mea~urements indicate that 4.3 amide protons within this 14-mer peptide are slowed by interaction o~ the ~6 antibody-with hemoglobin. A synthetic peptide identical to residues 1-14 o~
the hemoglobin B chain (Bl-14) was synthesized, tritium labeled by proton exchange, and subjected to graded digestion with carboxypeptidase-P (see Figure 6).
Similar studies were per~ormed with hemoglobin o~-exchanged a~ter interaction with ~121 monoclonal (Figure 3). Three pepsin-generated peptides were ~ound to bear tritium label (Figure 3, panel B). A~ter second ~;m~n.~ion HPLC separation in TFA-containing solvents these peaks were similarly resolved ~rom cont~m;n~nts, se~uenced, and ~ound to be hemoglobin polypeptides ~1-14, ~113-128, and ~15-31. In prel~m~n~ry proton counting studies, approximately two ~121 monoclonal-slowed protons are present in each of these three peptides.
The position o~ these peptidic regions in the ~olded hemoglobin tetramer are shown in Figure 5. The ~6 monoclonal labels six amide bonds which are present on an externally disposed segment o~ the ~olded hemoglobin molecule (~ chain amino acids 1-14) which includes the previously characterized target epitope o~ this monoclonal (~6-9) (51). The ~121 monoclonal labels a total o~ approximately six protons which, though present on the non-contiguous regions o~ the linear amino acid sequence o~ hemoglobin are seen to be sur~ace disposed and located in close proximity to each other in the ~olded hemoglobin molecule, and include the hemoglobin ~ chain 121 residue.

Ma~pinq of hemoqlobin-haptoqlobin interaction sites: When hemoglobin binds to haptoglobin it is known that the hemoglobin molecule contacts haptoglobin through three non-contiguous peptidic regions which consist o~ hemoglobin ~ chain 121-127, ~
11-25 and ~ 131-146 (52,53). We there~ore anticipated that pepsin cleavage o~ hemoglobin labeled at haptoglobin interaction sites would display between 2 and 10 radiolabeled peptides. We there~ore per~ormed our haptoglobin studies at a higher level o~
resolution, accomplished by collection o~ a larger number of HPLC
- ~ractions ~see Figure 4~. Under these conditions, labeled hemoglobin analyzed without a period o~ o~ exchange demonstrates W O97/41436 PCT~US96/06011 greater than 33 discernable radiolabeled peaks (Figure 4, panel B), which again correspond to the optical density tracing (Figure 4, panel A). Labeled hemoglobin o~-exchanged in the presence o~ haptoglobin produces 7 specifically radiolabeled peaks (Figure 5 4, panel C) which are not present i:E hemoglobin is o~ exchanged r in the absence of haptoglobin (Figure 4, panel D). These results indicate that this technology works well with a receptor-like ligand interaction system as complex as that o~ hemoglobin with haptoglobin.

Solvent E~ect Synthetic hemoglobin ~1-14 peptide was tritium-labeled at all peptide amides by proton exchange, and aliquots o~ labeled peptide subjected to 0~C HPLC analysis as in Figure 1 except that a range o~ solvent pH's were utilized as indicated below. The percent o~ original peptide-bound tritium that remained bound to the peptide under each HPLC condition was then determined.
E_ A solvent B solvent 2.1 0.115~ TFA in water 80~ A~N, 20~ H2O,0.1~ TFA
2.7 50 mM P04, pH 2.7 80~ ACN, 20~ 50mM P04, pH 2.7 3-5 50 mM P04, pH 3.5 80~ ACN, 20~ 50mM P04, pH 3.5 4.0 50 mM P04, pH 4.0 80~ ACN, 20~ 50mM P04, pH 4.0 Tritium retention was about 57~ ~or TFA (ph 2.1), 46~ ~or P04 (ph 2.7), 34~ ~or P04 (ph 3.5), and 14~ ~or P04 (ph 4.0).

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Claims (11)

1. A method of characterizing the binding site involved in the binding of a binding protein of known or determinable amino acid sequence to a binding partner which comprises a) providing a complex of the binding protein and the binding partner wherein one or more amide positions of a binding site of said binding protein are labeled with heavy hydrogen, said positions being, as a result of the complexation of said binding partner, not freely accessible to solvent, said labeling being substantially greater at said positions than at the amide positions which are freely accessible to solvent;
(b) dissociating the binding protein from the binding partner and fragmenting the protein, or a binding fragment thereof, to obtain a plurality of fragments, differing in heavy hydrogen content;
(c) substantially completely separating and purifying the fragments;
(d) determining which purified fragments are labeled;
(e) progressively degrading each purified, labeled fragment to obtain a series of residual subfragments of progressively smaller size, and quantifying the amount of heavy hydrogen label associated with each subfragment; and . (f) correlating the amount of heavy hydrogen label of the subfragments with the amino acid sequences of their source fragmetns, thereby localizing the particular amide positions of the binding protein that had been labeled with heavy hydrogen and thus further characterizing the binding site of said protein, wherein steps (b) through (e) are performed under conditions wherein the heavy hydrogen label is substantially retained at the labeled amide hydrogen positions of the binding protein.

2. The method of claim 1, wherein said complex of step (a) is provided by:
(i) contacting the binding protein with a heavy hydrogen-labeled solvent for an "on-exchange" period sufficient for substantially each of the exposed peptide amide hydrogen atoms of said protein to be replaced, in a detectable number of molecules of the protein, by heavy hydrogen;

(ii) forming a complex of the binding protein and its binding partner, wherein as a result of said binding some of said heavy hydrogen atoms become less accessible to solvent; and (iii) contacting said complex with an essentially unlabeled solvent containing normal hydrogen atoms for an "off-exchange" period sufficient for substantially all of the still exposed heavy hydrogen atoms to be replaced by normal hydrogen atoms, but where at least one heavy hydrogen atom is retained which, in the absence of said binding partner, would have been replaced by a normal hydrogen atom.

3. The method of claim 1, where said complex of step (a) is provided by:
(i) contacting the binding partner with a heavy hydrogen-containing solvent for an "on exchange" period sufficient for substantially complete (equilibrium) exchange of the exposed peptide amide hydrogen atoms of said protein with heavy hydrogen from the solvent;
(ii) separating and isolating the heavy hydrogen-labelled binding partner from the solvent under conditions which under which the heavy hydrogen label is substantially retained;
(iii) contacting the isolated, heavy-hydrogen-labelled binding partner, with a solution of binding protein that is initially free of solvent heavy hydrogen, under conditions such that complex formation spontaneously takes place between the initially unlabelled binding protein and the heavy hydrogen-labeled binding partner; and (iv) allowing heavy hydrogen transfer to occur from the binding partner to the portion of the binding protein that is occluded from efficient interaction with solvent by virtue of its interaction with binding partner;
whereby amide hydrogens of the binding site are more heavily labeled than amide hydrogens which are hidden from solvent as a result merely of allosteric changes occuring in the binding protein as a result of binding the binding partner.

4. The method of any of claims 1 to 3 wherein the label is tritium and the presence or amount of label on a fragment or subfragment is determined by radioactivity measurements.

5. The method of any of claims 1 to 3 wherein the label is deuterium and the amount of label on a subfragment is determined by measuring the mass of the subfragment.

6 The method of claim 5 wherein a tritium label is also used and the labeled fragments are identified by radioactivity measurements.

7. The method of any of claims 1-6 in which the separation is performed by two sequential separations under different conditions, preferably at two different pHs, each within the range 3.0 -2.1; such as at pHs 2.7 and 2.1.

8. The method of any of claims 1-7 in which the degradation of the fragments in step (e) comprises exposure of the fragments to an acid resistant carboxypeptidase, such as carboxypeptidase P, Y, W, or C, or to pentafluoropropionic acid anhydride.

9. The method of any of claims 1-8 wherein the binding protein initially features one or more disulfide bonds, and the method comprises disrupting said bonds, prior to the fragmentation of step (b), under conditions under which the tritiated or deuterated label is substantially retained at peptide amide hydrogens of the binding protein, e.g., by reaction with a water soluble phosphine.

10. The method of any of claims 1-9 where, in step (b), before said fragmentation, the protein is denatured, preferably with guanidine thiocyanate to render it more susceptible to fragmentation, and it is maintained in a susceptible state compatible with enzymatice fragmentation, preferably by dilution with guanidine HCl.

11. The methods of any of claims 1-10 wherein the fragmentation, or a denaturation preparatory to fragmentation, is carried out in a solvent such that the pH for minimization of hydrogen exchange is substantially higher than that of a purely aqueous solution, such as a solvent which is 5-20% water and the remainder a nonaqueous polar solvent such as acetonitrile or dimethyl sulfoxide, or a polyol such as glycerol.