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We have previously shown that an antigenic site in native lysozyme resides around the disulphide bridge 30-115 and incorporates Lys-33 and Lys-116 and one or both of Tyr-20 and Tyr-23. These residues fall in an imaginary line circumscribing part of the surface of the molecule and passing through the spatially adjacent residues Tyr-20, Arg-21, Tyr-23, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. The identity of the site was confirmed by demonstrating that the synthetic peptide Tyr-Arg-Tyr-Gly-Lys-Asn-Arg-Gly-Phe-Lys (which does not exist in lysozyme but simulates a surface region of it), and an analogue in which glycine replaced Tyr-23, possessed remarkable immuno-chemical reactivity that accounted entirely for the expected reactivity of the site in native lysozyme. Tyr-23 is not part of the site, and its contribution was satisfied by a glycine spacer. The novel approach presents a powerful technique for the delineation of antigenic (and other binding) sites in native proteins and confirms that these need not always comprise residues in direct peptide linkage.

SUMMARY We have previously shown that the antibody response to mammalian myoglobins is under genetic control. In the present study we examined antibodies to sperm-whale, Atlantic bottlenosed dolphin, Black Sea dolphin, horse and badger myoglobins, raised in high responder strains of mice, to ascertain whether there is genetic control of antibody affinity to mammalian myoglobins. Using antisera of varying dilutions, the binding to 125I-labelled homologous myoglobins was studied by inhibition with homologous myoglobin over a wide range of inhibitor concentration in a modified Farr assay. The results indicated that there are no large differences between high responder strains of mice in the affinity of antibodies to mammalian myoglobins.

The molecular localization of the full antigenic profile for T-cell recognition of a complex multi-determinant protein antigen has not to date been accomplished. Previously, this laboratory has introduced a comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein. This strategy depends on the synthesis of a series of overlapping peptides that together account for the entire structure of a protein. Such a strategy has been applied, in this report, to the delineation of the continuous sites of T-cell recognition of sperm whale myoglobin. Thirteen peptides, accounting for the entire protein chain, were synthesized and subsequently examined in vitro for their ability to stimulate lymph node cells from myoglobin primed DBA/2 (H-2d) mice, a known high responder. This strategy has enabled for the first time the localization of the full profile of the protein regions which contain the sites of T-cell recognition. Three regions gave a high response (one being immunodominant and coinciding with antigenic, i.e. antibody binding, site 4 of myoglobin). At least three regions appear to coincide with previously known antigenic (antibody binding) sites. Noteworthy is the finding of regions that are recognized by T-cells but to which no detectable antibody response is directed.

We have been able to isolate clones of sperm whale muscle myoglobin (Mb)-reactive T cells from (C57BL/6 x A/J)F1 [(B6A)F1] mice. Four types of clones were isolated, distinguished by their patterns of recognition of Mb cyanogen bromide (CNBr) fragments and antigen presenting cell (APC) requirements. Individual T cell clones proliferated in response to one of three CNBr fragments of Mb. Dose-response curves of all clones were identical for native Mb and the appropriate fragment. T cell clones reactive to fragment 1-55 did not proliferate in response to peptide 15-22 (a peptide that binds to serum antibody directed against 1-55). These data support previous findings suggesting differences between antigen recognition by T and B cells, i.e., T cells may not recognize antigen in its native conformation and/or T and B cells may recognize distinct epitopes on the same antigen. Using T cell clones to analyze genetic control of responsiveness to Mb, we found that certain (B6A)F1 T cells recognize Mb presented by low responder strain APC. Thus, genetically determined low responsiveness in this case is probably not due to failure of APC function. We also found that responsiveness to certain Mb epitopes mapped to the I-A subregion whereas others mapped, via gene complementation, to the I-A and I-E subregions. We found no examples of responsiveness mapping to the I-C subregion and suggest an alternative explanation for previous reports mapping genetic control of responsiveness to certain Mb determinants to I-C.

This paper reports the antigenicity of the fusion region of the influenza virus hemagglutinin (HA). Two peptides, comprising the fusion region (residues 1-11 of the HA2 part of HA) of strain A and strain B influenza virus, were synthesized and their abilities to bind rabbit, goat, and human anti-influenza antibodies were determined. In addition, 30 anti-HA monoclonal antibodies were examined for their ability to bind the synthetic peptides. In quantitative immunoadsorbent titrations, the two peptides bound considerable amounts of antibodies in rabbit and goat antisera against virus or HA of the A or B strain as well as in several human sera from patients recovering from influenza A. Of the 30 anti-HA monoclonal antibodies, 5 bound completely and 4 bound partially to the peptides. Antibodies were raised in rabbits against the peptides by immunizing with peptide-bovine serum albumin conjugates or with the free peptides. Anti-peptide antibodies were bound by HA and by the intact virus of the respective strain. However, these antisera failed to exhibit significant virus neutralizing activity. In contrast, the monoclonal antibodies that reacted with these peptides inhibited viral infectivity. The results clearly show that residues 1-11 of HA2 represent an important antigenic site on influenza virus.

Rabbit antibody was prepared to a derivative of human hemoglobin A 0 in which the two reactive sulfhydryl groups were substituted with N -ethylmaleimide. This antibody reacted completely with hemoglobins A 0 , A 1 , S 0 and a disubstituted parahydroxymercuribenzoate derivative of A 0 , suggesting that these sulfhydryl groups are in an immunogenically. silent region. Hemoglobins F 0 and A 2 and derivatives of A 0 with increasing amounts of parahydroxymercuribenzoate cross reacted with the antiserum. Globin, α-chains, and β-chains gave little reaction.

Conformational studies have been carried out on e-prototoxin and e-toxin from Clostridium perfringens , type D, by ORD and CD measurements. Also, any effect on the conformation, due to binding with e-toxin, of the peptide cleaved from e-prototoxin on tryptic activation of the latter was studied. The present work showed that e-prototoxin and e-toxin possess high amounts of β-conformation. The activation of e-prototoxin is accompanied by a conformational change since significant differences were observed in the ORD and CD parameters of e-prototoxin and e-toxin. The conformational change accompanying activation can be observed only if the cleaved peptide is removed or if measurements were carried out in a solvent that will not favor its binding to e-toxin.

The preparation of globin complexes with zinc metaloporphyrin, protoporphyrin IX and ferriheme dipyrid-4-ylpropyl ester is described and their physicochemical properties investigated. Reconstitution of hemoglobin from globin and resynthesized heme (i.e. from ferric iron and protoporphyrin IX) gave a preparation having conformational parameters and immunochemical behavior identical with those of the native protein. Hemoglobin derivative prepared with protoporphyrin IX showed a great increase in the Stokes radius which was accompanied by an enhancement of the antigenic reactivity of the derivative (relative to native hemoglobin). Similar increase in antigenic reactivity was obtained with hemoglobin containing heme dipyrid-4-ylpropyl ester, altough only relative slight changes in conformational paramaters were observed. The Stokes radius of the hemoglobin derivative prepared with zinc metalloporphyrin was greatly increased and reacted poorly with antisera to hemoglobin.

Structural similarities have been determined by mapping of the peptides of electrophoretically and chromatographically homogeneous myoglobins from human, monkey (Maccacus rhaesus), horse, camel, beef, lamb, goat and sperm whale. The amino acid compositions of myoglobins from monkey, camel, beef, lamb and goat have been carefully determined. Monkey Mb comprised 152 amino acid residues corresponding to a molecular weight of 17 557. Camel Mb had 153 residues and a molecular weight of 17 561. Beef and lamb myoglobins each had 153 residues and their molecular weights were 17 693 and 17 735, respectively. Goat Mb had 152 amino acid residues and its molecular weight was 17 594. Maximum structural similarities were between monkey Mb and human Mb; camel Mb and horse Mb; and lamb and beef Mb resembled goat Mb most closely. The antigenic cross-reactions of all the present eight myoglobins with antisera against human Mb, horse Mb, beef Mb, goat Mb or sperm whale Mb were determined. These results were also supported by absorption experiments on these sera. No cross-reaction was obtained between antisera to sperm whale Mb and the other seven myoglobins. With antisera to human Mb, monkey and camel myoglobins showed the highest and almost identical cross-reactions (75–83%). Horse Mb cross-reacted only partially (25 and 32% with the antisera tested). The other myoglobins did not react. With antisera to horse Mb, monkey and human myoglobins showed equal reactivities (30–33%) and the reactivity of camel Mb was only slightly higher (37 and 40% with the two sera tested). The other myoglobins did not react. With antisera to goat Mb, lamb and beef myoglobins showed equal reactivities (90–91%). The other five myoglobins did not react. With antisera to beef Mb, a stronger cross-reaction (87 and 90%) was obtained with lamb Mb than with goat Mb (76 and 72% with these two sera, respectively). The other five myoglobins did not react. These results together with those from absorption experiments, were discussed in relation to structural similarities of these eight myoglobins. The results show that in globular proteins, similarity in antigenic structure is not necessarily linearly related to similarity in sequence. The anamolies observed can be explained only in terms of the effect of differences in conformation.

A peptide comprising the sequence 1–31 in sperm-whale myoglobin (Mb) was isolated as one of the peptides from the specific cleavage of Mb at arginine peptide bonds. This peptide had previously been shown (Singhal and Atassi, 1971) to contain the single antigenic reactive region in the entire sequence 1–55. This was previously narrowed down to fall within (but not including all of) sequence 8–30. Carboxymethylation of peptide 1–31 with iodoacetic acid yielded a homogeneous derivative (CM 1–31) which has modified specifically at histidines 12 and 24. One histidine was carboxymethylated at position 3 in the imidazole ring and the other histidine was carboxymethylated at both position 1 and 3. Peptide 1–31 and CM 1–31 had identical ORD and CD parameters, clearly indicating that the modification did not result in a change of the peptide conformation in solution. This ruled out any conformational effects on the immunochemical behavior of the derivative. The antigenic reactivities of peptide 1–31 and CM 1–31 were quantitatively identical when determined with three different antisera to Mb. From previously reported work and the present results, the antigenic reactive region in the first third of the Mb molecule (sequence 1–55) has been shown to be located within (but may not include all of) sequence 13–23. Also, most likely glycine 23 will not participate in interaction with antibody.

Lysozyme from hen egg white, which is a “tight” ( i.e. proteolytically inaccessible, disulfide-containing) protein, was rendered accessible to tryptic hydrolysis at all arginine peptide bonds by reversible blocking of the amino groups with citraconic anhydride. After deblocking it was possible to effect complete cleavage at all the lysine peptide bonds, thus yielding all the tryptic peptides of lysozyme (termed (Arg, Lys)-peptides) without rupturing the disulfide bonds. It was highly significant that (Arg, Lys)-peptides inhibited strongly (85–89%) the reaction of lysozyme with its antibodies. The reactive fragments in the (Arg, Lys)-peptides were identified mainly as the three disulfide-containing tryptic peptides. These comprised sequences 22–33 (Cys 30–Cys 115) 115–116; 62–68 (Cys 64–Cys 80) 74–96 (Cys 76–Cys 94); and 6–13 (Cys 6–Cys 127) 126–128. The approach employed here provides a procedure by which the antigenic structure of tight proteins with disulfide bonds can now be studied without rupturing the disulfide bonds. Also, it affords another technique by which correct disulfide pairing may be determined.

Cleavage of sperm whale myoglobin at arginine peptide bonds has been accomplished by tryptic hydrolysis of the protein after blocking the amino groups reversibly with citraconic anhydride. Peptides comprising the sequences 1 to 31, 46 to 118, and 119 to 139 were isolated and studied by optical rotatory dispersion and circular dichroism. In the free state, each peptide was much less helical than expected from its conformation in the intact protein. Thus Peptide 1–31 which, in the native protein, has 30 out of 31 residues in a helical configuration (i.e. 97% helical) was only 33% helical (from b0 values) in the free state. Helicity increased appreciably (to 54%) in 62% methanol. The longer Peptide 46–118, which is 62% helical in the intact protein, was 38% helical in the free state and helicity of this peptide was increased to about 50% in 62% methanol. This is a long peptide with an elaborate mode of folding which makes the contribution of long range interactions quite feasible. Peptide 119–139 occupies a region in the native protein which is 71% helical. However, its helical content in the free state (15%) was quite low, mainly due to minimal contribution of long range interactions and to some loss of contribution of short range interactions since it carries only a portion of a helix (helix H). Although helicity of this peptide increased (to 31%) in 62% methanol, it was still much lower than the corresponding value for the other two peptides in the same solvent. These studies show that the conformation of each of these peptides is less helical in the free form than expected from its location and configuration within the intact protein. Helicity increases as the probability of stabilization of folding by long range interactions improves. Also, the results indicate that two separated fragments of a helix will not be able to achieve a high degree of helicity. Furthermore, proteins may not start to assume native conformation during biosynthesis until virtually or entirely completed, depending on the extent of contribution to long range interactions (in the complete protein) of the yet unsynthesized portion.

From considerations of polar, non-polar and hydroxy amino acid contents for several hemoglobins and myoglobins, it has been possible to point out composition differences that clearly differentiated the myoglobin group from the hemoglobin group. Within the same hemoprotein group, constant and characteristic composition tendencies could be pointed out that were typical of a particular class in the evolution ladder. Therefore, hemoglobins from the pisces were distinct in their composition characteristics from mammalian hemoglobins. Similar distinctions were also found in myoglobins. Composition characteristics were discussed in relation to structure, function and evolution of the hemoprotein groups. From this treatment it was deduced that the β-chain (rather than the α-chain) represents the primitive chain. Amino acid composition characteristics of hemoglobin in the order primates revealed certain tendencies which could be correlated with the phylogenetic classification and the recent amino acid sequences reported by others.

Modification of the arginine residues in lysozyme with cyclohexanedione has been carried out under two different conditions. The first derivative (CHD-Lysoz I) was prepared by reaction in 0·1 N NaOH. However, it was found that control lysozyme preparations subjected to these reaction conditions lost all enzymic activity, although their antigenic reactivity was not affected. To avoid this complication, a second derivative (CHD-Lysoz II) was prepared by reaction in 0·1 M triethylamine. In fact, lysozyme pretreated with this solvent retained entirely its enzymic activity and its antigenic reactivity. In both CHD-Lysoz I and CHD-Lysoz II, 10 arginine residues were modified. The two derivatives possessed identical conformations, as revealed from their optical rotatory dispersion and circular dichroism behaviors and from the availability of the disulfide bonds to reduction and susceptibility to chymotryptic attack, but were greatly unfolded relative to lysozyme. The two derivatives had no enzymic activity and very little antigenic reactivity with antisera to lysozyme. Conversely, with antisera to CHD-Lysoz I, lysozyme reacted only partially (60 per cent), and these sera showed appreciable (28 per cent) cross-reaction with CHD-human serum albumin. Interpretation of the enzymic and immunochemical data was complicated by the fact that too many (10) arginine residues were modified and that appreciable conformational changes were present. Preparation of a derivative with a least number of modified arginine residues was therefore desirable, and this was afforded by reaction of lysozyme with phenylglyoxal (PG-Lysoz). In PG-Lysoz, one arginine residue was modified and it was located at position 61. The derivative had identical optical rotatory dispersion and circular dichroism parameters with those of lysozyme. However, minor conformational changes were present, and these were only revealed by the slight increase in disulfide reducibility (0·26 bonds) and by a small increase in susceptibility to tryptic attack (from 0·16 bonds in lysozyme to 0·97 bonds in PG-lysozyme). The enzymic activity of PG-Lysoz was suppressed slightly (to 83 per cent) but the pH optimum was unaltered. With antisera to lysozyme, PG-Lysoz showed equal antigenic reactivity to that of lysozyme. Conversely, with antisera to PG-Lysoz, the reactions of lysozyme and PG-Lysoz were identical. Also, the two proteins had equal reactivities with antisera to CHD-Lysoz I (58–60 per cent relative to homologous antigen). It was, therefore concluded that arginine-61 is not located in an antigenic site of lysozyme. Also, the findings demonstrate that not all conformational changes will influence antigenic reactivity. The change in the latter will be dependent on the protein and on the nature of the conformational reorganization.

Apomyoglobin was modified specifically at the two methionine residues by reaction with β-propiolactone. The myoglobin derivative prepared by recombination of modified apomyoglobin with ferriheme possessed spectral and sedimentation properties that were similar to those of the native protein. Also the conformational parameters were identical. With antisera to the native protein modified, recombined myoglobin and recombined controls showed equal antigenic reactivities. Similarly equal antigenic reactivities were obtained with antisera to the modified, recombined protein. The latter reaction could not inhibited with an excess of the carboxyethyl sulfonium salt of methionine. The results confirm that the methionine residues (at positions 55 and 131) are not located in antigenic reactive regions in myoglobin.

1. Crystalline myoglobin was isolated from the skeletal muscle of the finback whale and fractionated, in its cyanmet form, into nine components (I-IX) by chromatography on CM-cellulose. Also in the cyanmet form, it was resolved into six components by electrophoresis on starch gel. Correspondence between the electrophoretic and chromatographic components was determined, and interconversion between components revealed by chromatography and electrophoresis. 2. The chromatographic myoglobin components were homogeneous in the ultra-centrifuge. Molecular weights of certain components were determined by means of sedimentation equilibrium and by gel filtration on Sephadex G-100. Values from these two methods corresponded to the minimum molecular weight calculated from the iron content. 3. The spectral properties of the chromatographic components were investigated in the visible and the ultraviolet ranges. 4. The major components of finback-whale myoglobin and sperm-whale myoglobin showed almost identical spectral, electrophoretic and chromatographic behaviours, but had different infrared spectra. The infrared spectra of the corresponding apoproteins were almost identical. 5. Rabbit antisera to sperm-whale myoglobin component X cross-reacted with finback-whale myoglobin components V, VI and VII only about 30%. 6. The major chromatographic components of finback-whale myoglobin have identical amino acid compositions. The polypeptide chain contains 151 amino acid residues and its molecular weight is 17504. 7. The N-terminal end of the chain is: [Formula: see text] Amino acids released from myoglobin by the action of carboxypeptidase A at different intervals were determined.

Reduction of carboxyl groups in lysozyme and myoglobin was investigated employing two alkylboranes with differing degrees of steric requirements with the aim of achieving steric control of the direction of hydroboration. Disiamylborane, a dialkylborane with large steric requirements, enabled the specific reduction of accessible glutamic acids and unhindered end-chain carboxyl groups. On the other hand, 9-borabicyclo[3,3,1]nonane, a bicyclic dialkylborane with appreciable hindrance, exhibited good selectivity for glutamate and unhindered C-terminal carboxylate groups with only marginal reduction of β-carboxyl groups of aspartic acid. In addition to their usefulness in specific chemical modification, these reagents may also prove to be valuable conformational probes.

1. The preparation and characterization of manganese, iron, cobalt, nickel, copper and zinc metalloporphyrins is described. Ferrihaem was also esterified with pyrid-4-ylpropanol and the derivative characterized as the diester. 2. Complexes of these various porphyrins, as well as protoporphyrin IX, with apomyoglobin were formed and the resulting artificial myoglobins characterized. 3. Very little complex-formation was obtained with nickel, cobalt and manganese metalloporphyrins and apomyoglobin. 4. Myoglobin prepared with copper metalloporphyrin was immunochemically identical with native ferrimyoglobin. All the other artificial myoglobins were less reactive to varying degrees. 5. The changes in antigenic reactivities were attributed to conformational reorganization caused by the different co-ordination tendencies of the various metals or by the modification of the side chains of the haem.

Conformational investigations have been carried out on derivatives of lysozyme in which tyrosines 20 and 23 were nitrated (NT2-lysozyme), or in which the nitrotyrosine residues had been reduced to aminotyrosine (AT2-lysozyme). Also, a derivative modified at the 6 tryptophan residues by reaction with 2-nitrophenyl sulfenyl chloride (NPS6-lysozyme) was studied. In optical rotatory dispersion measurements, NT2-lysozyme and AT2-lysozyme showed equal rotations at the negative 233-nm minima and at the positive 199-nm maxima. Also, they had identical b0 values. These two derivatives were more rotatory than lysozyme. On the other hand, NPS6-lysozyme showed an appreciable degree of unfolding evidenced by a decrease of all its optical rotatory dispersion parameters. Measurements of the reduced molar ellipticities showed that the present three derivatives, like lysozyme, give a negative circular dichroism band at 208 nm and a shoulder around 220 nm. Results were in agreement with optical rotation measurements. The rotatory behavior of lysozyme and the derivatives showed no change in the pH range 7 to 3. The conformational changes revealed by optical rotatory dispersion and circular dichroism measurements were also shown by increase in disulfide reducibility. Both NT2-lysozyme and AT2-lysozyme exhibited appreciable disulfide reducibility (one bond) relative to lysozyme (0.03 bond), and the great unfolding in NPS6-lysozyme was also confirmed by the large increase in accessibility of its disulfide bonds (2½ bonds). Changes in conformation obtained on binding of each of these proteins with sodium dodecyl sulfate were monitored by disulfide accessibility. It was shown that NT2-lysozyme and AT2lysozyme assume ‘relaxed’ conformation with identical disulfide accessibility (two bonds). The conformation of lysozyme also became ‘relaxed’, although to a lesser extent than the tyrosyl derivatives, on binding with sodium dodecyl sulfate. In contrast to these, NPS6-lysozyme, assumed a ‘constrained’ conformation on binding with sodium dodecyl sulfate. It was concluded that conformational changes take place upon modification of the tyrosine or tryptophan residues and that NT2-lysozyme and AT2-lysozyme had identical conformations. From these results and the previously reported immunochemical behaviors of these derivatives, it may be concluded that one (or both) of tyrosines 20 and 23 is located in an antigenic reactive site in lysozyme. The results also indicated that, although the antigenic reactivity of native proteins is highly influenced by changes in conformation, not every conformational change will exert an effect on the antigenic reactivity. This should be dependent on the protein and the nature of the conformational change.

The reaction of amino acids with Raney nickel has been carefully studied and the conditions most suitable for specificity determined. Two different grades of Raney nickel have been compared. One grade was prepared by addition of sodium borohydride to nickel acetate and the other was W 6 Raney nickel. Reaction was studied at pH 5.0, 7.0 and 8.0 at 40°. The most efficient conditions were at pH 7.0 and 40°. Cystine was completely converted to alanine in 4 min. No appreciable differences were found in the rates of the desulfurization of methionine which were extremely low. At pH 7.0 and 22° or lower, the reaction showed a high degree of specificity for cystine which was completely desulfurized in 12 min (at 22°) while methionine was essentially unchanged even after 10 h of reaction. The reaction has been applied for the specific desulfurization of cysteine (or cystine) residues in hemoglobin, lysozyme and α-lactalbumin, respectively.

Acylation of myoglobin (Mb) in the presence of 10 molar excess of TGA gave a heterogeneous reaction product which showed four major components by gel electrophoresis. Chromatography on CM-cellulose yielded four electrophoretically homogeneous tetramethyleneglutaryl-Mb components (TG-MbI, TG-MbII, TG-MbIII, TG-MbIV). The number of lysine residues acylated agreed well with the increase in electrophoretic mobility. TG-MbIV migrated like MbX and corresponded to residual unmodified MbX. The locations of the modified lysine residues in the derivatives were: TG-MbI, lysines 98, 140 and 145; TG-MbII, lysines 98 and 140; TG-MbIII, lysine 98. Absorption spectra of the four components were quantitatively identical with those of MbX. No conformational changes existed in TG-MbII or TG-MbIII as determined from measurement of molecular parameters and from ORD and CD studies. Slight conformational changes were observed in TG-MbI. Immunochemical studies with antisera to MbX showed that, with a given antiserum, TG-MbI, TG-MbII and TG-MbIII had equal antigenic reactivity which was 15–20% lower than the reaction of MbX with that antiserum. It was, therefore, concluded that lysine 98 is an antigenic reactive region in Mb. On the other hand, lysines 140 and 145 were clearly not part of a reactive region in Mb. From these findings and previously published results, it was possible to narrow down further a previously located antigenic reactive region in Mb so that it will now fall within sequence 146–151.

Haptoglobin binding to haemoglobin and its isolated alpha- and beta-chains was studied by use of a highly sensitive solid-phase radiometric assay. As expected, adsorbents of haemoglobin bound 125I-labelled haptoglobin more efficiently than did adsorbents of the alpha-chain. However, unexpectedly, adsorbents of the beta-chain were found to be essentially identical with those of the alpha-chain in their ability to bind haptoglobin. These results demonstrate, unequivocally, the ability of beta-chains to bind to haptoglobin, and indicate that this assay is particularly convenient and useful for studying haptoglobin interactions with haemoglobin and its alpha- and beta-chains.

Several years ago, this laboratory introduced a comprehensive strategy for the systematic localization of all the continuous sites on a protein that are involved in B- and T-cell recognition. The strategy depends on the synthesis of consecutive overlapping peptides that together account for the entire protein chain. Using this approach, the full submolecular profile of continuous regions on hen egg lysozyme recognized by T cells (T sites) were localized. Four major T-cell recognition sites, three of which were subject to individual genetic control, were localized in the six mouse strains examined. In addition to these four continuous T sites, T-cell recognition of lysozyme also involved the three previously defined discontinuous antibody binding sites as demonstrated with lysozyme-specific long-term T cell cultures. Contrary to a long held impression, T-cell recognition, therefore, is not restricted only to sequence features, but can also be directed to protein discontinuous surface areas of high conformational dependency. More recently, we have examined in two mouse strains the proliferative response to peptides and to native protein of lymph node cells from mice primed with synthetic overlapping peptides either individually or as a mixture. It was found that the pattern of T-cell recognition observed after priming with peptides differs from that obtained when the native protein is used as the immunogen. If antigen processing proceeds via fragmentation, then only those regions containing T sites would be expected to be effective in priming for a T-cell response to the intact protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The BALB/c myeloma protein ABPC48 (A48) binds the polysaccharide bacterial levan; its Vh is encoded by a gene derived from the VhX24 family. This antibody has been shown to crossreact idiotypically with the phosphorylcholine-binding BALB/c myeloma protein MOPC167 whose Vh shares homology with A48 from residues 32–44. We have synthesized a peptide corresponding to residues 32–44 of the Vh encoded by a germline gene of the VhX24 family. Anti-peptide antisera from rabbits were purified by affinity chromatography with peptide or intact antibody. Several myeloma proteins and monoclonal antibodies with varying degrees of homology to the peptide have been analyzed for reactivity with purified rabbit antibodies in solid-phase RIA. We observed that the specificities within rabbit antisera are heterogeneous, and that purification with antibody versus peptide yields preparations containing different specificities, albeit demonstrably peptide-related. We also show that injection of mice at birth with small amounts of purified rabbit antibodies can affect the magnitude of the response to bacterial levan and the expression of A48 idiotopes in that response.

Recently we have shown that the major antigenic sites of bovine serum albumin exhibit functional equivalence progessively increasing with the time at which antibodies are obtained after the first immunization. Analysis of our recent immunochemical findings and the known covalent structure of bovine serum albumin have enabled us to predict the locations of five antigenic sites of bovine serum albumin. The predicted locations were synthesized, and immunochemical studies with late-course antisera showed them to constitute antigenic sites of native bovine serum albumin.

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.

The fragmentation of native bovine serum albumin by trypsin has been studied in aqueous solution under various conditions with regard to the yield and size of the fragments obtained. From a partial tryptic hydrolysate at pH 8.2 (40 degrees, 1 hour), a homogeneous fragment was isolated in high yield by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose. The molecular weight of the fragment by gel filtration on calibrated Sephadex G-100 columns and by sodium dodecyl sulfate electrophoresis was 22,500. After reduction of the disulfide bonds followed by alkylation of the resultant thiol groups with iodoacetamide, the fragment retained homogeneity by disc electrophoresis and its molecular weight remained unchanged, indicating that it was composed of a single polypeptide chain. From its amino acid composition, sequence of the first 20 residues, and actions of carboxypeptidases A or B, it was unequivocally assigned to positions 377-571 in albumin. The inhibitory activity of the fragment was 90 to 93% towards the immune reaction of the protein with the IgG fraction of the antisera. The IgGfraction accounted for 96% of the total antibody activity in the antisera. An immunoabsorbent of fragment 377-571 removed 89 to 95% of the antibody to albumin. A fluorescent derivative of the fragment, which retained full immunochemical activity, was found to bind 2 mol of antibody/mol of peptide. The disulfides in peptide 377-571 were essential for its immunochemical reaction because the latter was entirely abolished upon reduction and S-alkylation of the disulfides. Since this fragment comprised only a third of the albumin molecule, but accounted for 90 to 95% of its antigenic reactivity, the results indicated that native albumin carries identical repeating antigenic reactive sites.

We have previously localized and synthesized twelve antibody recognition sites on influenza virus hemagglutinin (HA). These peptides correspond to exposed surface areas in the 3-D structure of HA. Using intact X31 virus as the immunogen, we have determined the recognition of these synthetic peptides by proliferative T-helper lymphocytes (ThL), delayed type hypersensitivity (DTH), and cytotoxic T-lymphocytes (CTL) responses. The responses to the individual determinants in each of these immune compartments were under separate Ir gene control. Conversely, using the peptides as immunogens, we have determined the ability of various peptide-specific antibodies (in outbred mice) and ThLs (in H-2k, H-2d, H-2s and H-2b mice) to recognize intact virus. Whereas most of the peptides primed the mice for an anti-peptide proliferative ThL response, only very few of these cross-reacted with the virus. The identity of the peptide(s) eliciting virus cross-reactive ThLs varied with the strain. The importance of antibody, ThL, CTL and DTH responses in protection against viral infection and in vaccine design is discussed.

By using the known antigenic structure of sperm-whale myoglobin previously determined in this laboratory and the X-ray co-ordinates for the myoglobin molecule, we have calculated the nearest-atom distances between each of the residues of the antigenic sites and all the other amino acids of the myoglobin molecule. These calculations have enabled us to identify the nearest-neighbour residues to each of the residues in the five antigenic sites, and which thus describe the immediate molecular environment of the sites. The influences of chemical changes or replacements in these environmental residues on the binding capacity of an antigenic site, when considered together with replacements directly in the antigenic sites, are expected to account for the major effects and will be extremely useful in explaining the cross-reactions of myoglobins from various species. However, it is stressed that the analysis has limitations due to the qualitative estimates of the effects, the influences of substitutions of once-removed or even at more distant locations (especially when they are cumulative) and finally the influences of any conformational re-adjustments when these occur as a result of the replacement(s).

1. We have previously shown that an antigenic site (site 1) in native lysozyme resides around the disulphide bond 6-127 and, by classical synthesis of nine disulphide peptides, the antigenic site was accurately narrowed down to the structure Cys((6))-Arg((14))-[Cys((6))-Cys((127))] -Gly((126))-Arg((128)). Only a few residues on this disulphide peptide were proposed to be involved in the reactivity with antibody. However, this lacked direct verification and the role of Arg-128 remained uncertain. 2. In the present work, several peptides were designed and synthesized by the surface-simulation concept devised in our laboratory. These enabled the precise definition of the site as well as the investigation of its conformational and directional requirements. 3. The results showed that the antigenic site (site 1) is made up of the spatially contiguous surface residues: Arg-125, Arg-5, Glu-7, Arg-14, Lys-13. The surface-simulation synthetic peptide Arg-Gly-Gly-Arg-Gly-Glu-Gly-Gly-Arg-Lys (which does not exist in native lysozyme, but copies a surface region of it) accounted entirely for the maximum expected reactivity of the site (i.e. about one-third of the total antigenic reactivity of lysozyme). An immunoadsorbent of the peptide also removed about one-third of the total lysozyme antibodies. 4. The antigenic site exhibited restricted conformational freedom. The achievement of the full reactivity of the site by surface-simulation synthesis requires the appropriate choice of spacer separation between its reactive residues. The surface-simulation synthetic site exhibits the same mono-directional preference (Arg-125 to Lys-13) for the rabbit and goat antisera so far tested. The site describes a line which encircles a part (3.01 nm in C((alpha))-to-C((alpha)) distance from Arg-125 to Lys-13) of the surface of the molecule.

Recently, we have localized and confirmed by synthesis the regions within which reside five major antigenic sites of bovine serum albumin and proposed that the remaining sixth major antigenic site (antigenic site 4) was localized within subdomain 3 (fragment 115-184) of albumin. In the present work, antigenic site 4 was localized to fall around the disulfide bond 166-175 by synthesis and immunochemical reactivity of the region 162-179. The synthetic 18-residue peptide was shown to bind substantial amounts of antibodies from early (38-day) and late (398-day) anti-albumin antisera from two rabbits. As much as half the total anti-albumin antibodies could be bound by the peptide from the late antisera. It was concluded that antigenic site 4 resides within, but may not include all of, the region 162-179 of albumin.

A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.

This paper reports the localization of the regions on the beta-chain that are recognized by T cells from mice immunized with haemoglobin. The 14 overlapping peptides encompassing the entire beta-chain were examined in vitro for their ability to stimulate lymph-node cells from haemoglobin-primed B10.D2 (H-2d) and SJL (H-2s) mice. Several regions of the molecule (T sites) were found to stimulate haemoglobin-primed lymph-node cells. This strategy has enabled the localization of the full profile of T-cell recognition of the beta-chain by these mouse strains. Some of the regions that stimulated T cells appeared to coincide with those recognized by antibodies (i.e. B cells). It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has other sites that are recognized exclusively by T cells and to which no detectable antibody response is directed.

Six regions (T sites) of myoglobin (Mb) were found by a comprehensive synthetic strategy to stimulate Mb-primed lymph-node cells. To define precisely the N-terminal boundary of the immunodominant T site (residues 107-120) with site-specific T-cell clones and to determine the effects of peptide size on their stimulation, two sets of peptides were employed. In one set, the peptides were elongated to the left from His-113 by one-residue increments of the Mb sequence. The other set represented an identical stepwise elongation by one-residue increments of the Mb sequence, but which were extended by additional unrelated (‘nonsense’) residues to a uniform size of 14 residues. Examination of the proliferative responses of eight T-cell clones, derived from Mb-primed DBA/2 (H-2d) or SJL (H-2s) mice, revealed a dramatic non-specific size requirement. In every clone, the longer nonsense-extended peptides achieved maximum stimulating activity at a lower optimum peptide dose than its natural-sequence, but shorter, analogue. In addition, slight (one-residue) differences in the N-terminal boundaries among the clones was observed. Thus, the fine specificity of each clone was mapped to the region from residue 111 or 112 to about residue 120 of Mb, which coincides with the site of B-cell recognition and resides in a small discrete surface region of the protein chain.