CA2094798A1 - Method of mapping polypeptide ligand binding regions - Google Patents

Abstract

METHOD OF MAPPING POLYPEPTIDE
LIGAND BINDING REGIONS

ABSTRACT

A method is provided for mapping regions of a polypeptide exhibiting selective affinity for a chemical species. The method comprises the steps of digesting the polypeptide with an exoprotease, substantially free of endoprotease activity, to produce a series of partially digested polypeptides of different molecular weight, but a common undigested terminus. The position of the targeted region in the primary structure of the polypeptide is defined by determining the molecular weight of the lowest molecular weight species exhibiting the selective affinity.
The method has particular utility for mapping antibody-binding or receptor-binding epitopes.

Description

METHOD OE' MAPPING POLYPEPTIDE
LIGAND BINDIMG REGIONS
Field of the Invention This invention relates to a rapid method of mapping polypeptide ligand binding regions. More particularly, this invention is directed to a method utilizing partial enzymatic digestion of polypeptides and subsequent separation and hybridizing/labeling of the product polypeptide partial hydrolysates to locate the approximate position of regions in the primary structure of the polypeptide that exhibit selective affinity for other chemical species.

Backqround and Summary of the Invention Knowledge of~the mechanisms and the sites of molecular interactions of polypeptides and proteins (hereinafter referred to genera]ly as "polypeptides") with biologically active chemical species, includiny peptides, polypeptides, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), DNA/RNA analogs, carbohydrates, phospolipids and other chemical species of biological significance can provide insight into the biolQgical activities of such chemical species. Such knowledge can be an invaluable resource for medical research and development, including ; particularly, drug design. Examples of common molecular interactions of biological significance are the high ; affinities and the resulting selective bonding of antibodies to their respective target antigens, cellular receptors to their target chemical species, and enzymes to ; their target substrates.
Antibodies are proteins produced by vertebrates as a defense against infection. Each antibody contains a unique binding site that exhibits selective affinity for at least a portion of the molecular structure of the antigen ,, ~ ; , , ; ~ ~ ': , ;

s~ 79~

that induced its production. Those unique molecular structures on an antigen that are affinity~targeted by the antigen binding site on an antibody are referred to as antigenic determinants or epitopes. Most antigens present a multiplicity of antigenic determinants on their surfaces.
In the study of antibody-antigen interaction, it is often important to determine exactly what region (epitope) of the antigen structure is interacting with (binding to) a particular antibody and what region on the antibody structure is interacting with the antigen.
Traditional approaches to antibody epitope mapping all require amino acid sequence information at some stage of the characterization protocol. Protein footprinting relies on the ability of the antibody to protect the antigenic site from random proteolysis or chemical modification, but the protected peptide must invariably be sequenced to identify the epitope. Limited antigen digestion followed by immunoblotting may also identify a cross-reactive fragment, but again the fragment must be sequenced to locate its position in the parent antigen. While comparison of antibody affinity for a series of nearly homologous proteins can often correlate differences in specificity with known amino acid substitutions, such closely related, previously sequenced protein families are not common, and considerable effort is required to generate them artificially by mutagenesis of a cloned antigen. Alternatively, competition between antigen and synthetic or natural peptides for binding to the antibody can often reveal the targeted epitope, but again the peptide must be sequenced to determine its location in the antigen's primary structure. In a few cases, epitopes have been assigned without sequence information to an enzyme' 5 active site based on the antibody's ability to inhibit catalytic activity, but even in that case caution :;

.. : . . - . . ; , , - : : ~

must be exercised to ensure that the antibody does not inhibit enzyme activity by a noncompetitive mechanism.
Due to the expense and delay involved in conventional methods of epitope mapping, there exists a strong need for a less cumbersome method of epitope mapping. More generally, there is a need for a means for mapping regions of polypeptides primary structure that exhibit selective affinity for certain chemical species.
Accordingly, it is one object of this invention to provide a method for locating sites or regions (in polypeptide primary structure) of associative interaction between a polypeptide and a ligand comprising a second chemical species.
It is another, more particular object of this invention, to provide a rapid method for identifying the location of antibody binding antigenic determinants in the primary structure of polypeptides.
It is still another object of the invention to provide a method for mapping regions of the primary structure of an exoprotease digestible polypeptide which exhibit sel~ctive affinity to a chemical species (ligand) by partially digesting the polypeptide with an exoprotease and determining the molecular weight of the lowest molecular weight species still exhibiting said selective affinity.
In still another embodiment of this invention there is provided a kit for mapping regions or epitopes in the primary skructure of a polypeptide.
Those and other objects are realized in accordance with this invention which is directed particularly to a protocol that allows identification of the approximate position of an epitope or other region in the primary structure of a protein exhibiting affinity to a chemical species without requiring the tedious procedure of sequencing said epitope or region. Generally, the method ,, ~
.. . .
:; -, , , . ,: . , ' ' '"' ',~ :

. .

in accordance with this invention comprises the steps of subjecting the targeted polypeptide to denaturing conditions to initiate unfolding of the polypeptide and thereafter digesting the polypeptide with an exoprotease substantially free of endoprotease activity to provide a mixture of a first set of polypeptide hydrolysate species containing the region of the polypeptide primary structure that exhibits selective affinity to the chemical species and a second set of polypeptide hydrolysate species that do not contain said region. By determining the moleculax weight of the lowest molecular weight polypeptide species conkaining the chemical species-binding region, one can locate or map that region relative to one or the other termini of the parent polypeptide structure. The procedure can thus be applied to define regions of polypeptide primary structures that exhibit affinity either for other polypeptides or for other chemical species, including biologically active drug substances and other biologically significant chemical species including, but not limited to, peptides, DNA, RNA, DNA/RNA analogs, carbohydrates, lipids and phospholipids.
Additional objects, features, and advantages in the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the preferred embodiments exemplifying the best mode of carrying out the invention.

Detailecl Description of the Invention In accordance with one embodiment of this invention, the location of a region on a polypeptide which exhibits a selective affinity for a chemical species is determined by a procedure that includes the partial digestion of the polypepkide with an exoprotease. To ensure efficient exoprotease diyestion of the polypeptide, ; 35 the polypeptide is preferably subjected to denaturing `:
,, :. .: . ;
,.':,,' ' ~' ' ' " :

;' ~

~: , '

2 ~ rl 9 ~

conditions sufficient to initiate unfolding of the polypeptide, thus ~nhancing exoprotease access to the terminal end of the polypeptide. Polypeptide denaturants well known to those of ordinary skill in the art include urea, aliphatic amines, guanidine hydrochloride, ionic and non-ionic surfactants, and heat. Each of these denaturants or denaturant conditions can function separately or in combination with other denaturant excipients, for example, art-recognized disulfide-bond-reactive thiols to initiate or otherwise enable the unfolding of the target polypeptide. The potency or stringency of the denaturiny conditions is dependent on both the denaturant selected and the concentration of the denaturant. The potency of the denaturing conditions is optimally selected to be within a range that can initiate unfolding of the polypeptide, without significantly inhibiting exoprotease activity.
After the at least partially denatured polypeptide has been subjected to exoprotease digestion for a time sufficient for the exoprotease to initiate digestion, the potency or stringency of denaturing conditions in the digestion medium can be reduced without a adverse affect on the digestion process due to refolding of the polypeptide. One preferred means of reducing the denaturant stringency in the digestion medium is to carry out the digestion in an aqueous medium in contact with a dialysis membrane to allow the denaturants and amino acid digestion products to diffuse from the medium during the digestion process. High concentrations of cleaved amino acid residues can work to inhibit exoprotease activity and 30 their removal from the medium allows polypeptide digestion ~`
to continue at a more or less constant rate.
The exoprotease used to digest the polypeptide can be selected from any of the exoproteases well known to those of ordinary skill in the art. The exoprotease must be substantially free of endoprotease activity and capable .

, . . :
,: ' ,. , . ~
..

,:, , ' . ' 2~l7~8 of cleaving amino acids only from one end, either the ; carboxy-terminus or the amino-terminus, of a polypeptide.
Further, the exoprotease should exhibit its proteolytic activity in the presence of denaturing conditions, sufficient to initiate unfolding of the target polypeptide and it should preferably effect a more or less constant or consistent rate of digestion.
Carboxypeptidases and aminopeptidases comprise two classes of exoproteases. Carboxypeptidases cleave amino acids sequentially from the C-terminus of a polypeptide, while aminopeptidases cleave amino acids sequentially from the N-terminus of a polypeptide. In a preferred embodiment of this invention, the exoprotease used to di~est the polypeptide being assessed in accordance with the present invention is selected from the group consisting of carboxypeptidases and aminopeptidases.
Again, care must be taken to ensure that the polypeptide and the exoprotease are substantially free of endoprotease activity. The polypeptide is digested for sufficient amount of time to provide a mixture of a first set of polypeptide hydrolysate species containing the chemical species-affinity-exhibiting-region and a second set of polypeptide species hydrolysate not containing the affinity-exhibiting region.
The next step for mapping of the affinity-exhibiting region of the polypeptide is the determination of the molecular weight of the lowest molecular weight species of the polypeptide hydrolysates ;; still capable of exhibiting selective affinity for the chemical species. One preferred means of making such a determination includes the step o~ chromatographically resolving the mixture of the partially digested peptide species on the basis of polypeptide species molecular weight. After the polypeptide hydrolysate species have been chromatographically resolved, they can be fixed in , ,~

, ~''' ' , ~ " , ' '' " '' ' ' ' ~ , , ',1,. .. .
.. . .

~, , " : , ~
, , . . .
, : ~

2~A7~

their separated state, for example, by transfer to a nitrocellulose or nylon membrane or by other means well known to those of ordinary skill in the art. The separated polypeptide hydrolysate species can then be contacted with the chemical species to which the original whole polypeptide exhibited a selective affinity under conditions which said selective affinity can be exhibited by still existing or intact affinity-exhibiting regions of the polypeptide.
A polypeptide may exhibit a selective affinity for a chemical species due to the conformational shape of the affinity exhibiting region. These conformational ~; affinity-exhibiting regions consist of discontinuous amino acid sequences that are in close spatial proximity to one another in the secondary and tertiary structure of the protein. When the discontinuous amino acid sequences are separated by large intervening sequences, a partially digested polypeptide will likely cease to exhibit affinity for the chemical species once the first most terminal discontinuous amino acid sequence of the affinity exhibiting region is cleaved. Thus, without prior knowledge that the affinity exhibiting region contains component amino acid regions separated by large intervening sequences, the interpretation of the exoprotease map could be misleading. To avoid this problem, and to verify the accuracy of the mapping, the polypeptide can be subjected to two separate mapping procedures, each in line with that described above, except one mapping procedure is carried out using an aminopeptidase as the exoprotease, while the other mapping procedure is carried out using a carboxypeptidase as the exoprotease. In this manner the affinity exhibiting region will be mapped in reference to distance from the C-terminus and from the N-terminus. If the affinity-exhibiting region is a conformationally dependent affinity-exhibiting region, (i.e., based on the ' , : -: ,' , ' '~ ; .
. ,,, ~ :, .
"' ~ , ..
"

7 ~ ~

cooperation of discontinuous "partial epitop~s" in-the primary polypeptide structure~ each mapping procedure will indicate differing locations for the affinity exhibiting region.
Compositions for mapping regions of the primary structure of a protein which exhibit selective affinity for a chemical species in accordance with this invention can be provided in the form of a reagent kit. The kit comprises ; an exoprotease and an endoprotease inhibitor, and optionally a protein denaturing composition selected to provide a denaturant stringency sufficient to initiate protein unfolding but insufficient to denature the exoprotease. Thus an epitope mapping kit in accordahce with this invention can include either one or both of a carboxypeptidase and an aminopeptidase and an endoprotease inhibitor. Preferably the kit contain~ one or more additional components such as a denaturant solution of predetermined stringency (or components for the preparation thereof), dialysis membrane tubing, and a solution to quench or stop the digestion reaction.
One advantage of the method of this invention is that it does not require purification of the targeted polypeptide containing a selective affinity exhibiting region. As long as the polypeptide is free of endoprotease ~ 25 activity, the presence of other contaminants should not j interfere with performance of this method. Thus, the regions of the primary structure of an exoprotease digestible polypeptide which exhibit a selective affinity to a chemical species can be mapped relative to either terminus of the polypeptide. The method comprises the steps of partially digesting the polypeptide with an ;; exoprotease, where the exoprotease and the polypeptide are substantially free of endoprotease activity to provide a mixture of polypeptide hydrolysate species having a common undigested terminus and di~ferent molecular weights. The ,~

:- , . ... . . ..

, ! - i i ' ' ' ~ '':' ' ' ~

. .
: . ' ~

polypeptide hydrolysate species are then chromatographically separated on the basis of the species molecular weight. The separated polypeptide species are then contacted with the subject chemical species under conditions in which selective affinity can still be exhibited by still existing or intact affinity exhibiting regions of the polypeptide. By determining the molecular weight of the polypeptide hydrolysate species exhibiting selective affinity for the chemical species, the location of the affinity exhibiting region on the polypeptide can be readily determined.
In accordance with one preferred embodiment of this invention, the position of an antibody-binding epitope on a polypeptide antigen is determined. The polypeptide antigen is digested with a one or more carboxypeptidases to provide a mixture of polypeptide hydrolysate species, each containing the same N terminus but different degrees of truncation at the C-terminus. After electrophoretic separation and immunoblotting, the epitope-containing polypeptide hydrolysate fragments are identified by art-recognized techniques utilizing labelled antibodies capable of providing a visual signal of the hydrolysates still containing the intact epitope. The molecular weight of the smallest molecular weight N-terminal fragment still capable of selectively interacting with the antibody is then identified. The molecular weight of the polyp~ptide hydrolysate correlates to the number of amino acids subunits contained in the polypeptide. Thus, the distance - of the epitope from the N-terminus of the target polypeptide antigen can be directly ascertained from the immunoblot without need for sequence information.
In a preferred embodiment, an antibody-binding epitope on a polypeptide can be located relative to either terminu~ of the polypeptide by a rapid method that does not require amino acid sequence determination. The method " . , ~ , . -. , , ~

'' ~9~798 defines the location of the epitope in terms of itsdistance generally in molecular weight units from the N-terminus or C-terminus of the intact protein. When a protein is diyested by either an aminopeptidase or a carboxypeptidase for a length of time insufficient to complete the digestion, there is produced a series of polypeptide hydrolysates differing only in length (i.e., molecular weight). If a polypeptide is digested with a carboxypeptidase, all the hydrolysate species in the series will have the same N-terminus; the hydrolysate species will differ only in the number of amino acids removed from the carboxyl end. In at least a portion the polypeptide hydrolysates generated from the partial digestion reaction, digestion will have proceeded through the location of the epitope. These species are no longer be capable of exhibiting selective affinity to the epitope-targeting antibody. The polypeptide hydrolysates having the epitope still intact can exhibit selective affinity to the antibody. Determination of the smallest polypeptide hydrolysate (lowest molecular weight) exhibiting selective affinity to the antibody identifies the location of the epitope relative to the end terminus of the intact protein.
; Highly folded protein structures are often inefficiently cleaved by exopeptidases. Therefore, to obtain a relatively continuous distribution of polypeptide fragments (partial hydrolysates), it is important to promote at least some degree of polypeptide unfolding of ; such structures prior to digestion. This can be accomplished by subjecting the polypeptide to denaturing conditions effective to initiate unfolding. Protein ~-~ denaturants are well known in the art and can be used alone or in combination at different concentrations to attain a predetermined denaturing stringency or potency. One preferred way of initiating unfolding (denaturing) a protein is to contact the protein in a denaturing solution .

, :
.: :
:, ', :

~9~7~8 containing, for examplP, urea, dithiothreitol, and methylamine. The dithiothreitol is included in the solution to disrupt intramolecular disulfide bonds that could otherwise interfere with exoprotease digestion. The exoprotease is typically added to the resulting solution of the at least partially unfolded polypeptide. After an initial one hour digestion under denaturing conditions, the denaturants can be gradually removed by dialysis without compromising the ability of the exoprotease to continue digestion. Thus the exoprotease digestion step can be conducted in a dialysis tubing. Typically the dialysis tubing has a low molecular weight pore size ranging from 1000-3000 ~, that will allow denaturants and released amino acids to diffuse out of the dialysis tubing while retaining small protein fragments. Reducing the concentration of the denaturants and the amino acids in the reaction medium allows the exoprotease to function in a more efficient manner. The dialysis tubing is usually suspended in the same buffer used for the denaturing/digestion process, but without (or lower concentrations of) denaturants. The reaction is preferably supplemented with fresh enzyme periodically during the digestion process to ensure a constant digestion rate.
;~ One critical feature of this invention is the elimination of any endopeptidase activity in the digestion reaction medium. Even small amounts of contami.nating endopeptidase in the digestion medium will invalidate the results, because analysis of the data assumes the digestion is only occurring from one terminus of the polypeptide.
The presence of contaminating endopeptidases will generate ~ smaller peptide fragments containing the targeted - interactive site than would be yenerated with exopeptidase alone. The production of smaller epitope-containing polypeptide hydrolysates will give an anomalous indication of the distance of the epitope or interactive site from the ,~

, ~ ,. .

, 7 ~ ~

terminus of the protein. One method of eliminating or minimizing contaminating endopeptidase activity in the digestion reaction is to include an inhibitor of endopeptidase activity such as ~2-macroglobulin. ~2-5 ~acroglobulin is a potent inhibitor of virtually all endopeptidases.
Digestion of the polypeptide is continued for a period of time sufficient to generate a mixture of polypeptide hydrolysate species having a common undigested 10 terminus and different molecular weights. Essentially there is produced a set of hydrolysate species that still contain the target region and a set of hydrolysate species that do not still contain the target region. The reaction can be terminated by boiling the digestion mixture or by 15 adding a strong denaturant. The resultant mixture of hydrolysate species are separated by chromatographic means according to molecular weight. One preferred means of chromatographically separating the polypeptide hydrolysate species utilizes electrophoresis through a gel matrix.
20 Typically, sodium dodecyl sulphate (SDS) polyacrylamide yel electrophoresis is used to separate the hydrolysate ~; species, but other art-recognized chromatographic means can also be utilized.
t The separated hydrolysate species are preferably 25 fixed in their separated state prior to assessing their affinity to the subject chemical species. One preferred ~- manner of fixing the hydrolysate species involves blotting the fragments onto a membrane such as nitrocellulose or a nylon filter. The re~ulting immunoblots can then be 30 hybridized with the chemical species, ~or example, an antibody capable of binding to the target epitope to determine the lowest molecular weight species containing the targeted reyion (epitope). Because high resolving SDS
polyacrylamide gels can often determine protein molecular 35 weights within 2,000 daltons, the present methodology :
~, ,, , :

.~

~.

2~rl9 allows mapping of regions exhibiting the selected a~finity in the primary structure of a polypeptide with the same degree of resolution.
Standard laboratory techniques can be used to label or visualize the hydrolysate species still exhibiting selective affinity to the chemical species. For example, to visualize which hydrolysate species still contain an antibody~binding epitope, the antibody that recc~gnizes the epitope (primary antibody) is first hybridized with the blot containing the transferred hydrolysate species. After unbound primary antibody is washed off the blot, a secondary antibody (an antibody capable of binding to the primary antibody) is hybridized with the blot, followed by washing to remove any unbound secondary antibody. The secondary antibody is selected to have an enzyme conjugated to it that is capable of reacting with a substrate to alter the substrate in a manner that can be visualized.
Preferred secondary antibodies include goat anti-mouse or ~ goat anti-rabbit IgG-horseradish peroxidase conjugates.
; 20 Thus addition of the substrate will provide a visual indication where the primary ant:ibody has bound and thereby identify which hydrolysate species still contain the target epitope.
More generally, a variety of methods well known to those of ordinary skill in the art can be utilized to ~ visualize chemical species that interact with the -~ immobilized polypeptide hydrolysate species. 'rhey include - but are not limited to radioisotope labeling, fluorescent labeling, and conjugating indicator molecules or enzymes capable of reacting with indicator molecules to the chemical species.
One common method for labeling a chemical species uses an avidin-biotin complex. The methods used to synthesize biotinylated chemical species, are well known in the art.
See, ~or example, E.A. Bayer and M. Wilcheck, The Use o*

'':

2 ~

the Avidin-Biotin Complex as a Tool in Molecular Biology, Methods of Biochemical Analysis, Vol. 26, pp. 5-9.

EXAMPLE ONE
Yeast carboxypeptidase Y and porcine pancreas carboxypeptidase B (From Calbiochem) were selected because of their resistance to denaturation by 6 M urea. Also, a mixture of the two proteases yielded a less interrupted digestion pattern than either carboxypeptidase alone. ~2-Macroglobulin immobilized on agarose beads was ~btainedfrom Boehringer-Mannheim. Nitrocellulose paper (0.2 ~m pore siæe) from Schliecher and Schuell, prestained low-molecular-weight markers from GIBCO BRL, electrophoresis reagents from Bio-Rad, and dialysis membranes (molecular weight cutoff ~2000) from Spectrum were obtained.
Antigen and Antibodies The 43,000 Da (residues 1-379) cytoplasmic domain of human erythrocyte band 3 protein (cdb3) was purified to homogeneity from fresh human blood. The polyclonal cdb3 anti-peptide antibodies, p32-34, p22 23, pl6-17, and pOO-O1 were raised in rabbits against synthetic peptides corresponding to residues 283-297, 189-203, 142-154, and 1-15 of the cdb3, respectively. The monoclonal antibodies, `~ mAb41-43 and mAb36-41 have been mapped to residues 360-379 and 317-359 of cdb3 previously using conventional epitope mapping techniques.
Carboxypeptidase Digestion Because even small amounts of contaminating endoproteases in the digestion mixture could invalidate the results (vide infra), it became necessary to take special ; precautions to inhibit such contaminants. For this purpose, both the antigen (1 mg/ml cdb3) and the carboxypeptidase mixture (1000 units/ml of carboxypeptidase Y and 100 units/ml of carboxypeptidase B) dissolved in digestion buffer (50 mM sodium citrate, 10% acetonitrile, ;

. .... . . ..

, , : ~ , : ' , ',: . ' , ,, ,, , , , . : ,:: . : :, : : : :
,:::: : . : .: ::
:, :. :,: :;:

2 ~

pH 6.0) were separately incubated for thirty (30) minutes at room temperatura with 0.1 volume of agarose beads containing immobilized ~2-macroglobulin, a potent inhibitor of virtually all endoproteases. After incubation, the heads were removed by centrifugation and an ali~uot of the carboxypeptidase mixture (10 units of Y plus 1 unit of B) was added to 1 ml of the cdb3 solution supplemented with 6 M urea, 10 mM dithiothreitol, and 20 mM methylamine (final concentration) to promote cdb3 unfolding. The unfolded cdb3 was allowed to digest for one (1) hour at room temperature, after which the mixture was transferred to ;-dialysis tubing suspended in digestion buffer supplemented with 1 mM dithiothreitol. The digestion mixture in the tubing was then treated again with ten (10) units of carboxypeptidase Y plus 1 unit of carboxypeptidase B and the digestion was continued in the dialysis bag for five (5) more hours. After this period and again six (6) hours ~ later the dialysis bag was opened and the cont~nts were ; treated with the same amount of the carboxypeptidase ~; 20 mixture. After a total of twenty-four (24) hours ~ digestion, the reaction was terminated by boiling (5 -; minutes) and the polypeptide hyclrolysate species were separated on a 15% Laemmli polyacrylamide gel and then transferred to nitrocellulose for three (3) hours at 240 mA. The resulting blots were blocked for fifteen (15) minutes with 4~ bovine serum albumin in blotting buffer (20 mM NaCl, pH 7.5) and incubated three (3) hours with ascites fluid containing monoclonal antibody diluted 1:500 in blotting buffer or with rabbit anti-cdb3 peptide antibody diluted 1:100 in blotting buffer. The nitrocellulose sheets were then washed with blotting buffer containing 0.05~ Tween 20 and incubated two (2) hours with goat anti-mouse or goat anti-rabbit IgG-horseradish peroxidase conjugate diluted 1:500 in blotting buffer. Blots were stained with 4-chloronaphthol.

,~

2~47~

Results Carboxypeptidases comprise a class of proteases that cleaves amino acids s~quentially from the C-terminus of a polypeptide. Polypeptide cdb3 (Mr ~3,000; 379 residues) was digested by the carboxypeptidase mixture into a series of different molecular weight peptides. When digestion was extended to thirty-six (36) hours or terminated prematurely at twelve (12) hours, a disproportionately large number of low- or high-molecular-; lo weight hydrolysate species, respectively, was obtained, suggesting the twenty-four (24) hour digestion period was roughly optimal for cdb3. The derived peptides were all found o contain the amino-terminal region of cdb3, as evidenced by their reactivity with pOO-Ol antibody. When stained with antibodies previously mapped by more tedious methods to relatively evenly spaced epitopes along cdb3, ~i the fragmentation mixture yielded a staining pattern that - differed for the different epitope-specific antibodies.
Thus, m41-43, which recognizes an epitope between resides 360 and 379, stained only the largest molecular weight ~` component of he digestion mixture corresponding to a band at Mr >35,000, but no fragments of smaller molecular weight.
The staining of p32-34 was terminated at M~ ~33,000, the staining of p22-23 at Mr 22,000, pl6-17 at Mr -17,000 and pOO-O1 near the bottom of the gPl. Thus, the digestion mixture in each lane was immunostained from the molecular weight position of the intact antigen to the approximate position of tne Ppitope and no further. By observing where the staining pattern terminated, a rough estimate of the 3G position of the antibody's epitope in the primary structure of the antigen was obtained. Importantly, when an impure mixture of the antigen and other proteins was trPated similarly, an analogous result was obtained as long as unwanted proteases did not contaminate the mixture.

, ;
: . . . .
: :,, ,, :~
,: : ., ,, , :
: .

2~79~

In developing this methodology, a number of significant obstacles were encountered that required modifications in the protocol. First, all commercial carboxypeptidases tested came contaminated with small amounts of endoproteases that cleaved the antigen internally. Proteolysis with such enzyme preparations generated fragments that were predictably stained by the above antibodies at anomalously low molecular weights. For example, the anti-peptide IgG, p32-34, stained blots of cdb3 digested with an unmodified mixture of commercial carboxypeptidases Y and B discontinuously from the molecular weight of the intact protein (Mr ~42,000) to ~16,000 Da. In contrast, when the carboxypeptidase mixture was pretreated with ~2-macroglobulin (a protein that inhibits all endoproteases without inactivating ` exoproteases), the staining pattern terminated at the expected molecular weight of 33,000 Da. Thus, the correct epitope of p32-34, located between residues 283 and 297 (i.e., 33,000 Da from the N-terminus) was correctly identified only when all endoprotease activity was eliminated.
A second obstacle arose from the tendency of the carboxypeptidases to balk at cleaving highly folded protein. However, if the antigen was first unfolded in 6 urea, 10 mM dithiothreitol, and 20 mM methylamine, a more continuous/complete digestion pattern was observed.
Therefore, to obtain a relatively continuous distribution of N-terminal cdb3 fragments, it was necessary to promote at least some degree of protein unfolding. After the initial one (1) hour digestion under denaturing conditions, the denaturants could be gradually removed by dialysis without comprising the ability of the proteinase mixture to continue cdb3 digestion. A dialysis membrane with a low-molecular-weight cutoff (M, ~2000) was necessary to avoid loss of the smaller protein fragments.

2~7~

Finally, because intrachain disulfide bonds interfere with carboxypeptidase digestion, the reaction mixture was always supplemented with dithiothreitol.
omission of this reagent l~d to appearance of major discontinuities in the cleavage pattern of cdb3. The digestion was also carried out mainly in a dialysis bag to allow the escape of released amino acids that at high concentrations inhibited the carboxypeptidases.
Discussion For many experimental applications of antibodies, ; a crude evaluation of the antibody's epitope on its antigen is sufficient. The present invention provides a relatively ~- simple method for identifying such epitopes in terms of their distance in molecular weight units from the N-terminus of the intact antigen. The most obvious advantages of this protocol are (i~ that no sequence in~ormation is required, and (ii) that the antigen need not ~- be pure. That is, as long as the antigen is not degraded by contaminating endoproteases, other polypeptides should not interfere with the map since they should not be visualized in the immunoblots.
Although the invention has been described in detail with reference to certain preferred embodiments, ~; variations and modi~ications exist within the scope and spirit of the invention as described and defined in the following claims.

,. .. .

' ' ': ' ':' ''' ' ,;" "
,: , , ', ' ' ,

Claims (27)

CLAIMS:

1. A method for locating or mapping an antibody-binding epitope on a polypeptide, said method comprising the steps of subjecting the polypeptide to denaturing conditions to initiate unfolding of the polypeptide, digesting the polypeptide with an exoprotease selected from the group consisting of a carboxypeptidase and aminopeptidases, said exoprotease being substantially free of endoprotease activity, to provide a mixture of a first set of polypeptide hydrolysate species containing said antibody-binding epitope and a second set of polypeptide hydrolysate species not containing said antibody-binding epitope, and determining the molecular weight of the lowest molecular weight polypeptide species containing said epitope.

2. The method of claim 1 wherein the exoprotease comprises a carboxypeptidase whereby the location of the epitope relative to the amino-terminus of the polypeptide is defined.

3. The method of claim 1 wherein the exoprotease comprises an aminopeptidase whereby the location of the epitope relative to the carboxy-terminus of the polypeptide is defined.

4. The method of claim 1 wherein the polypeptide partial digestion is conducted in the presence of an endopeptidase inhibitor.

5. The method of claim 4 wherein the endopeptidase inhibitor is .alpha.2-macroglobulin.

6. The method of claim 1 wherein the partial digestion process is carried out in an aqueous medium in contact with a dialysis membrane to allow amino acid digestion products to diffuse from the medium during the digestion process.

7. The method of claim 1 wherein the determination of the molecular weight of the lowest molecular weight polypeptide hydrolysate species containing the epitope includes the step of chromatographically resolving the mixture of partially digested polypeptide hydrolysate species on the basis of polypeptide species molecular weight.

8. The method of claim 7 wherein the polypeptide hydrolysate species are separated using SDS
polyacrylamide gel electrophoresis.

9. The method of claim 2 wherein the polypeptide is subjected to a second epitope mapping method the same as that of claim 2 except that an aminopeptidase is used instead of a carboxypeptidase to provide a second indication of the location of the epitope along the polypeptide.

10. A method for mapping a region of the primary structure of a polypeptide which exhibits a selective affinity to a chemical species, said method comprising the steps of subjecting the polypeptide to denaturing conditions to initiate unfolding of the polypeptide, partially digesting the at least partially unfolded polypeptide with an exoprotease selected from the group consisting of carboxypeptidases and aminopeptidases, said polypeptide and said exoprotease being substantially free of endoprotease activity, to provide a mixture of polypeptide hydrolysate species having a common non-digested terminus and different molecular weights, and determining the molecular weight of the lowest molecular weight polypeptide hydrolysate species that exhibits selective affinity to said chemical species.

11. The method of claim 10 wherein the chemical species is a compound selected from the group consisting of peptides, polypeptides, deoxyribonucleic acids, ribonucleic acids, carbohydrates, lipids, phospholipids, and other biologically active drug substances.

12. The method of claim 10 herein the determination of the molecular weight of the lowest molecular weight polypeptide hydrolysate species that exhibits selective affinity to the chemical species includes the step of chromatographically resolving the mixture of the polypeptide hydrolysate species on the basis of polypeptide species molecular weight.

13. The method of claim 12 wherein the polypeptide hydrolysate species are separated using polyacrylamide gel electrophoresis.

14. The method of claim 10 wherein the exoprotease comprises a carboxypeptidase.

15. The method of claim 10 wherein the exoprotease comprises an aminopeptidase.

16. The method of claim 10 wherein the polypeptide partial digestion is conducted in the presence of an endopeptidase inhibitor.

17. The method of claim 10 wherein the partial digestion process is carried out in an aqueous medium in contact with dialysis membrane to allow amino acid digestion products to diffuse from the medium during the digestion process.

18. A kit for mapping a region of the primary structure of a polypeptide which exhibits selective affinity for a chemical species, said kit comprising an exoprotease, a polypeptide denaturant composition, and an endoprotease inhibitor.

19. The kit of claim 18 wherein the exoprotease is substantially free of endoprotease.

20. The kit of claim 18 wherein the exoprotease is a carboxypeptidase.

21. The kit of claim 18 wherein the exoprotease is an aminopeptidase.

22. The kit of claim 18 wherein the endoprotease inhibitor is .alpha.2-macroglobulin.

23. The kit of claim 18 further comprising a dialysis membrane.

24. The kit of claim 24 further comprising a dialysis membrane.

250 A method for mapping regions of the primary structure of an exoprotease digestible polypeptide which regions exhibit a selective affinity to a chemical species said method comprising the steps of partially digesting the polypeptide with an exoprotease, said exoprotease and said polypeptide being substantially free of endoprotease activity, to provide a mixture of polypeptide hydrolysate species having a common undigested terminus and different molecular weights, and determining the molecular weight of the lowest molecular weight polypeptide hydrolysate species exhibiting selective affinity for said chemical species.

26. The method of claim 26 wherein the chemical species is a compound selected from the group consisting of peptides, polypeptides, deoxyribonucleic acids, ribonucleic acids, carbohydrates, lipids, phospholipids, and other biologically active drug substances.

27. The method of claim 26 wherein the chemical species is an antibody or a cell receptor polypeptide.