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Extensive chemical and immunochemical work in our laboratories has now culminated in the accurate delineation of the entire antigenic structure of myoglobin (Mb). These studies have all been reported. Five antigenic reactive regions have been accurately narrowed down, and were six or seven residues in size. In the present work, antibodies against MbX were prepared from two antisera, labelled with I 125 and adsorbed on Mb-Sepharose immunoabsorbent. The five antigenic reactive regions, prepared by organic synthesis, were each studied with regard to their abilities to displace specific antibodies from the Sepharose-Mb-antibody complex. Batchwise displacement with one reactive region only, followed by recovery of total elutable antibody, showed that of the five antigenic reactive regions only region 56–62 failed to elute specific antibodies in this fashion. The other four reactive regions each effected appreciable displacement of antibody, the extent of which depended on on the immunochemical reactivity of that region previously reported from solution immunochemistry. Lack of reaction of region 56–62 was attributed to steric hindrance on the immunoabsorbent, exerted by antibody molecules bound to the other reactive regions. The obstruction prevented approach of free region 56–62 to the corresponding antigen-antibody binding sites. This was confirmed by serial displacement studies with the five synthetic peptides. Removal in series of antibody molecules bound to the other four reactive regions of Mb first, followed by elution with peptide 56–62 enabled the displacement of specific antibodies by the latter peptide. The amount of displacement with a given immunoabsorbent agreed well with the expected reactivity of the region with that antiserum. Serial elution with five synthetic regions usually displaced about 81% of the total elutable antibody. Reabsorption of specific antibody, thus eluted by a giben reactive region, on Mb-Sepharose followed by serial elution with the five antigenic reactive regions showed that this antibody was displaced only by that particular reactive region. It was therefore concluded that in early course antisera, the five antigeniv reactive regions of Mb were immunochemically independent and showed no immunochemical interaction.

The two-disulfide peptide 62–68 (Cys 64–Cys 80) 74–97 (Cys 76–Cys 94) has been shown in previous reports in this series to carry a full antigenic reactive site of lysozyme. It was also shown that the reactive site required, for appropriate reactivity, one or both of tryptophans 62 and 63 and one or both of lysines 96 and 97. Also, some or all of the region 84–93 around Asp 87 was needed for achievement of full reactivity. When the contribution of the latter was obliterated, the reactivity of the site decreased (from about 28–33% down to 13–16%; Atassi et al ., 1976 a ). The integrity of the disulfides 64–80 and 76–94 was critical for bringing the various regions of the site into close proximity. In the present communication, the independent roles of the two trytophans and the two lysines were investigated by studying the immunochemistry of seven synthetic peptides. These peptides were designed to stimulate the sequences: 62–64 linked to 76–80 which is in turn linked to sequence 94–97. However, we have employed a novel and an unorthodox approach in that our synthetic peptides carried diglycyl segments instead of disulfide bonds. Also, the four peptide bonds in the central part of each synthetic peptide (designed to simulate the segment 76–80) were of the opposite direction to the natural sequence. In several of the peptides, tryptophan residues were substituted by phenylalanine. Peptides with one tryptophan, one phenylalanine or two phenylalanines at the amino end possessed comparable inhibitory activities which approximated the aforementioned expected value. When the peptide did not carry a tryptophan or a phenylalanine at the amino end, the inhibitory activity decreased drastically (down to 3–5%). Removal of one or two lysines on the C-terminal end of the peptides (but adding back two phenylalanines at the amino end to maintain size and contribution of that portion to immunochemical reaction) had an adverse effect on the immunochemical reaction. The decrease in inhibitory activity was greater on deletion of the last lysine than on deletion of the second lysine. These studies showed that only one tryptophan (residue 62) is part of the antigenic site in lysozyme, whereas both lysines are required for the reactivity of the site with one being more critical than the other. The results also indicated that diglycyl segments substituted with surprising

Previous work from this laboratory has shown that Arg-118 is part of an antigenic reactive region in myoglobin (Mb). In the preceding communication, we demonstrated that this reactive region also includes histidines -113, -116 and -119. This evidence was derived from immunochemical and conformational studies on chemical derivatives of the antive protein. The present communication presents independent evidence for the presence and accurate delineation of this reactive region obtained from studying the immunochemistry of synthetic peptides representing various parts of the region. Five peptides corresponding to Mb-sequences 108–120, 109–120, 112–120, 113–119 were synthesized, well purified and characterized. The reaction of these peptides with four different antisera to Mb demonstrates that the full immunochemical reactivity resided entirely in sequence 113–119 and that the part 108–112 and Pro-120 did not add to the immunochemical reactivity of the peptides. The reactive region, therefore, occupies the sequence 113–119 accurately, which is located on the end of helix G and part of the bend GH in native Mb. This completes the antigenic structure of Mb, and the latter is briefly outlined here.

Reaction of lysozyme with carbodiimide followed by coupling with glycine methyl ester (GME) or with histidine methyl ester (HME) resulted in the preparation of two derivatives. After column chromatgoraphy the two derivates were homogeneous by starch gel electrophoresis. Both derivatives were modified at aspartic acid 119 and at the carboxyl group of the end-chain leucine residue. However, the one derivative the two carboxyl groups were coupled to GME (GME2-Lysoz) and in the other, these two carboxyl groups were couple to HME (i.e. HME2-Lysoz). Each derivative suffered a dreastic loss in enzymic activity, most likely as a result of conformational changes which accompained the modification. HNE2-Lysoz showed a greater loss in lytic activity than GME2-Lysoz. With antisera to lysozyme, GME2-Lysoz possessed equal antigenci reactivity ot that of the homologous antigen, while HME2-Lysoz suffered an appreciable decrease in reactivity. Antisera were also raised against GME2-Lysoz and with these antisera, lysozyme and GME2-lysoz possessed equal antigenic reactivities. On the other hand, HME2-Lysoz showed lower antigenic reactivity. From these results and previously reported conformational studies, which showed very slight conformational changes in GME2-Lysoz and large changes in HME2-Lysoz, it was concluded that aspartic acid 119 and the C-terminal leucine residue are not essential parts of an antigenic reactive region in lysozyme.

Alkylation of myoglobin (Mb) in the presence of 10 M excess of ICH 2 COOH per histidine residue gave a heterogeneous reaction product which contained at least six components by disc electrophoresis. Chromatography on CM-cellulose yielded seven homogeneous carboxymethyl-Mb derivatives (CM-MbI—CM-MbVII). The electrophoretic mobility of each component agreed well with the number of carboxymethyl groups added. CM-MbVII migrated like MbX and corresponded to residual unreacted protein. The locations of the modifications in the derivatives were: CM-MbVI, histidine 119; CM-MbV, histidine 48 and 97; CM-MbIV, hisitidines 81 (or 82) and 116; CM-MbIII, lysine 145 and histidines 48 and 113; CM-MbII, histidines 48, 113 and 116; CM-MbI, valine 48, 113 and 116. Slight changes in absorption spectra of CM-MbI and CM-MbII were observed, while the spectra of MbX and the other components were quantitatively identical. Also, ORD and CD measurements indicated that some conformational changes were present in CM-MbI and CM-MbII and that no conformational changes existed in CM-MbIII—CM-MbVII. Immunochemical studies with antisera to MbX showed that, with a given antiserum CM-MbIII and CM-MbIV had equal antigenic reactivity which was 18–21 per cent lower than reaction of MbX with that antiserum. With CM-MbVI, the decrease in immunochemical reaction was not as large (11–13 per cent). It was concluded that histidines 113, 116 and 119 were part of one antigenic reactive region. The immunochemical reaction of CM-MbI and CM-MbII was 30–32 per cent lower than MbX. However, because these two derivatives suffered a conformational change, conclusions relating to antigenic structure were not based on these two derivatives. The immunochemical reactivity of CM-MbV was also lower (18–19 per cent) than the homologous reaction and the decrease was attributed to alkylation of histidine 97 in the previously delineated reactive region 94–99. The results also confirmed that valine 1, histidines 48 and 81 and lysine 145 were not part of a reactive region in Mb.

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.

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.

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.

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.

Rabbits were inhected in the hind foot pads with 5 mg sperm whale myoglobin (Mb) in complete Freund's adjuvant. At least 30 days later the draining popliteal lymp nodes were removed for the preparation of cell cultures. These cultures were challenged for the first 24 hr with various amounts of either Mb or synthetic Mb peptides. The medium collected after 24 hr of culture was employed for the assay of macrophage inhibitory factor (MIF). 14 C-leucine was added to the cells during 72–120 hr of culture to radioactively label newly synthesized antibody (to Mb). The culture medium collected at 120 hr was then utilized for the assay of radioactive antibody. Synthetic Mb peptides representing sequences 1–6, 15–22, 15–23, 16–22, 54–62, 56–62, 57–63, 94–100, 113–119 and 146–151 induced the Mb-primed lymph node cell cultures to synthesize antibody to Mb. Peptides from all of these regions—except 1–6-induced these cultures to produce MIF. The addition of a singe peptide to a culturel usually induced either antibody or MIF, not both. These results are discussed in terms of some of the possible molecular and cellular mechanisms of induction of these responses by these peptides.

Previous work from this laboratory on the antigenic structure of lysozyme has shown that an antigenic reactive site resides within, but may not include all of, sequence 62–68 (Cys 64-Cys 80), 74–97 (Cys 76-Cys 94). This two-disulfide peptide accounted for about a third of the total antigenic reactivity of native lysozyme. We had also previously shown that one or both of tryptophans 62 and 63 as well as lysine 96 (and a lesser contribution from lysine 97) are essential parts of the antigenic site which requires also intact disulfides to bring these two parts of the site together. The present work investigates the role of arginine 68 and aspartic acids 66 and 87. Activation of carboxyl groups with carbodiimide followed by coupling with histidine methyl ester yielded a homogeneous derivative modified at all free carboxyl groups and possessed identical ORD parameters to those of the unmodified peptide. However, the derivative retained only less than half of the immunochemical reactivity of the peptide. Reaction with phenylglyoxal resulted in the modification of arginine 68 as well as the N-terminal residues tryptophan 62 and asparagine 74 and one e-amino group. The derivative showed no change in conformational parameters but suffered a loss of about half the immunochemical reactivity relative to the unmodified peptide. Reaction with 1,2-cyclohexanedione, also resulted in the modification of arginine 68 and, in addition, the two tryptophans, the two lysines and the N-terminal asparagine 74 were modified. The derivative showed no change in conformational parameters but lost almost its entire inhibitory activity. Reaction with phenylglyoxal after protection of the free amino groups by citraconylation followed by removal of the protecting group, yielded a homogeneous derivative which was specifically modified at arginine 68 only. This derivative suffered no change in conformation or in immunochemical behavior relative to the unmodified peptide. From these studies it was concluded that one (or both) of aspartic acids 66 and 87 is part of the antigenic site, whereas arginine 68 is not located in the antigenic site on the peptide. Coordination of these and our previous findings, yielded a definition of the complex boundaries of the antigenic site in this part of the molecule.

Previous work from this laboratory on the antigenic structure of myoglobin (Mb) has shown that an antigenic reactive region exists within, but may not include all of, sequence 86–102 of Mb. Also, recently we have reported evidence that this reactive region is centered around histidine-97 and lysine-98. The present communication present independent evidence aimed at the accurate delineation of the reactive region by studying the immunochemistry of synthetic peptides corresponding to various parts of the region. Four peptides corresponding to sequence 92–100, 93–100, 94–100 and 93–99 were synthesized, exhaustively purified and characterized. The immunochemical interactions of the peptides were studied with four different antisera to Mb. With the antisera studied here, the presence or absence of Ser-92, His-93 or Pro-100 did not infleunce the immunochemical reactivity of the peptides. The reactive region, therefore, occupies the sequence 94–99 accurately. This is a small (six residue) surface location on the corner between helices F and G in native Mb.

The two subunits (α and p) of ragweed antigen E (AgE) were obtained by gel filtration of Citraconylated AgE on Sephadex G-75. The subunits had molecular weights of 25,000 and 13,900 respectively by disc electrophoresis in sodium dodecyl sulfate and, in amino acid analysis, gave the expected compositions for the α and β chains of AgE. The presence of IgE antibody to α and β subunits in sera from a pool of ragweed sensitive persons was tested by the radioallergosorbent test (RAST). After removal of the citraconyl masking groups, both the α and β subunits showed remarkable activity, in terms of ability to bind IgE antibody from a pool of ragweed sensitive persons.

Modification of the amino groups in lysozyme was carried out by guanidination, acetylation, succinylation or maleylation. The specificity of each reaction for amino groups was carefully determined and the enzymic and immunochemical properties of all derivatives studied. Reaction of lysozyme with 1-guanyl-3,5-dimethylpyrazole nitrate gave an electrophoretically homogeneous derivative in which five amino groups were guanidinated. No other amino acids were modified and the derivative exhibited no conformational changes and suffered no loss in enzymic activity, relative to native lysozyme. By acetylation of lysozyme with different amounts of acetic anhydride, two electrophoretically homogeneous derivatives were prepared. Both derivatives were acetylated at all seven amino groups, but one had seven and the other had all fifteen hydroxy amino acids esterified. Reaction of lysozyme with succinic or maleic anhydride resulted in electrophoretically heterogeneous preparations even when all amino groups were modified. There was also a non-uniform esterification of aliphatic hydroxy amino acids. The acetylated, succinylated, or maleylated derivatives showed small, but measurable conformational changes. The enzymic activity was completely eliminated upon acylation of four or more amino groups and this loss was attributed to changes in electrostatic interactions between the modified enzyme and the negatively charged bacterial cell wall. With antisera to lysozyme, the guanidinated derivative possessed full (100 per cent) antigenic reactivity, relative to lysozyme. The two acetylated preparations retained substantial, and comparable, antigenic reactivity with these antisera. In succinylated or maleylated derivatives, the electrophoretic heterogeneity was not accompanied by immunochemical heterogeneity. A considerable amount of cross-reactivity was retained, even when all amino groups were succinylated or maleylated. Partially or completely succinylated lysozymes were antigenic in rabbits and antibodies to the completely succinylated derivative exhibited (a) a specificity against lysozyme, (b) a specificity against antigenic regions containing succinyl groups and showing carrier specificity and (c) a haptenic specificity against succinyl groups attached to unrelated carrier proteins. From these findings it was concluded that retention of the charge of certain amino groups is essential for intact antigenic reactivity. Some amino groups (possibly four) and at least eight aliphatic hydroxyl groups are not located within antigenic sites of lysozyme. Furthermore, succinyl and maleyl groups are immunochemically distinct.