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The sequence of the alpha-chain of the acetylcholine receptor of T. californica has been determined by recent cloning studies. The integrity of the disulphide bond between Cys-128 and cys-142 has been shown to be important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulphide bridge in binding with acetylcholine. In the present work, a synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labelled alpha-bungarotoxin to the synthetic peptide. It was further shown that the free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulphide structure was essential for binding. Unlabelled acetylcholine also inhibited the binding of labelled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Torpedo receptor.

The determination of the entire antigenic structure of myoglobin has made it possible to focus attention on the molecular factors controlling and influencing immune recognition. Recently, using antisera raised against sperm-whale myoglobin (Mb) in six different host species and investigating their reactions with Mb from 15 species, we showed that the binding capacity of an antigenic site is influenced by substitutions within site residues as well as within the residues close (within 6.0 a) to the sites. Based on these effects it was possible to correlate, at least in qualitative terms, the expected effects of the substitutions in each Mb and its observed cross-reaction with antisera to sperm-whale Mb. In the present work, these correlations were tested using sperm-whale Mb antibodies raised in four inbred mouse strains and in which the amount of antibodies directed to each antigenic site was determined. The amounts of 125I-labelled antibodies that could be bound maximally be each of 12 Mb variants were determined. Because of genetic control, the response to some antigenic sites was not expressed and therefore permitted us to evaluate with a good degree of confidence the effects of amino acid replacements in various myoglobins upon the reactivity of the sites. The values of cross-reaction expected for each Mb variant from these considerations agreed well with the values found experimentally. The results confirm unambiguously that the major factors affecting the cross-reactions of proteins can be attributed to substitutions within the sites and within environmental residues of the sites.

The previously described peptide 62-68 (Cys 64-Cys 80) 74-96 (Cys 76-Cys 94) (Atassi, M.Z., Suliman, A.M. and Habeeb, A.F.S.A. (1975) Biochim. Biophys. Acta 405, 452-463), which accounted for about one-third of the total antigenic reactivity of native lysozyme, was isolated here with lysine 97 attached to it. The peptide was subjected to specific modification reactions in order to determine some of the residues which formed part of its antigenic reactive site. ORD measurements showed that the peptide was greatly unfolded in solution relative to its expected mode of folding within the intact lysozyme molecule. Modification of the two tryptophan residues in the peptide by reaction with 2,3-dioxo-5-indolinesulfonic acid provided a derivative which possessed similar conformational parameters to those of the unmodified peptide. However, the derivative retained only about half the immunochemical reactivity of the peptide. Succinylation of the amino groups afforded a derivative whose conformational parameters were identical to those of the unmodified peptide but in which half of the immunochemical reactivity was lost. Modification of the two tryptophan residues followed by succinylation of the amino groups resulted in almost complete loss of the antigenic reactivity, and the loss was not due to conformational differences. The antigenic reactivity of the peptide was also destroyed on removal of tryptophans 62 and 63, of sequence 84-93 from the loop 74-79 and of sequence 74-75 by chymotryptic digestion. From these and previous results it was concluded that the antigenic reactive site in this part of the lysozyme molecule incorporates one or both of tryptophans 62 and 63 as well as one or both lysines 96 and 97. The two disulfides 64-80 and 76-94 bring these two parts of the lysozyme molecule into a single reactive site. The intactness of the disulfides is essential for maintenance and reactivity of the site.

Determination of the T-cell recognition profile of Mb by the overlapping peptide strategy revealed that the protein has six T sites. Five of these coincide with the antigenic sites while one site was recognized exclusively by T cells and not by any detectable levels of antibody. Recognition of the synthetic T sites by protein-primed T cell lines or clones indicated that T cells display an unusual peptide size requirement beyond the essential contact residues of the T site. The antigen presentation of Mb has been examined with the significant advantage of knowing the full profiles of T- and B-cell recognition of this multi-determinant antigen. Significant differences in the patterns of T-cell recognition were observed following protein-priming as compared to peptide-priming. The absence of a clear relationship between these patterns of recognition presents a strong evidence against a mechanism of antigen presentation which is dependent on the generation of peptide fragments with the latter being the ‘presented’ species. From this new perspective, the protein molecule must be predominantly presented in its intact form.

Mice were immunized with 14 free (i.e. not conjugated to any carrier) synthetic peptides representing the entire human hemoglobin alpha-chain. Antibodies against each peptide were determined using solid phase radioimmunoassay, both with free peptides and peptides coupled to a protein carrier as the coating antigen. It has been demonstrated that large improvements in the ability to detect anti-peptide antibodies were achieved in some cases by precoating the assay wells with free peptides and in other cases by precoating with peptide-protein conjugates. Sodium carbonate buffer, pH 9.6, had a favorable effect on the coating of two of the free peptides when compared with phosphate-buffered saline, pH 7.2. The assay with the plates coated with optimum peptide form (free peptide or peptide-protein conjugate) was superior in the detection of antibody binding to 9 of the peptides when compared with the assay using chemically activated plates. The results suggest that the appropriate form (conjugated or free) and conditions for immobilizing small peptides to plastic supports are not universal but will have to be determined for each test peptide.

A comprehensive synthetic approach, previously developed in this laboratory, has been applied to systematically screen the entire extracellular part (residues 1-210) of the alpha chain of the Torpedo californica acetylcholine receptor (AChR) for the profiles of the continuous regions that are recognized by antibodies against free, or membrane-sequestered, AChR; the regions recognized by AChR-primed T cells; the regions that bind alpha-bungarotoxin and cobratoxin; and an acetylcholine-binding region. Eight continuous antigenic sites were localized in this part of the alpha chain by all of the antisera tested. The sites were independent of the host species from which the antisera were obtained and were also similar to antisera against the isolated pentameric AChR or against the membrane-sequestered AChR. Six regions were found to stimulate AChR-primed T cells (T sites). Three of the T sites coincided with regions recognized by antibodies. At least two T sites had no detectable antibody responses directed to them. Five toxin-binding regions were localized, and may constitute distinct sites or, alternatively, different faces in one (or more) sites. Some of these regions coincided with regions recognized by anti-AChR antibodies. One of the toxin-binding regions bound acetylcholine, and immunization with this peptide induced experimental autoimmune myasthenia gravis.

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.

The systematic localization of the full antigenic profile for T-cell recognition of a complex multivalent protein antigen has not been accomplished. Recently, this laboratory has developed a comprehensive strategy for the localization of all the continuous antigenic (as well as other binding) sites of a protein. The strategy depends on the synthesis of consecutive overlapping peptides that together systematically account for the entire polypeptide chain of the protein. This strategy was applied here for the localization of the 'continuous' T-cell recognition sites of hen egg lysozyme. Eight overlapping peptides encompassing the entire primary structure of lysozyme were synthesized and examined for their ability to stimulate in vitro proliferation of T cells from two high responder mouse strains (H-2k and H-2q) that had been primed with lysozyme. This approach enabled the identification of a full profile of in vitro active lysozyme peptides and the localization of four major T-cell recognition sites.

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.

Recent studies from this laboratory have found, contrary to a long-held belief, that synthetic peptides of a protein, as small as six residues, when immunized in their free form (i.e., without coupling to any carrier), elicit the formation of antibodies with submolecular binding specificities to preselected protein regions. These peptides could represent either the antigenic sites of the protein or surface regions that are not immunogenic when the intact protein is the antigen. In either case, the antibodies bind specifically to the intact protein, exclusively at the region used in immunization. Monoclonal antibodies with preselected specificities can also be produced by hybridoma technology from the spleens of the immunized animals. Furthermore, free synthetic peptides have been employed to generate T-cell lines and T-cell clones with specificities to preselected immunogenic locations and also to induce tolerance to such preselected locations. These fascinating breakthroughs indicate that free synthetic peptides may be used in the future as powerful tools in basic investigations and in therapeutic and diagnostic applications.

Bovine serum albumin was rendered accessible to tryptic hydrolysis at the arginine peptide bonds by reversible blocking of the amino groups with citraconic anhydride. The tryptic digest showed 83% inhibition of albumin reaction with anti-bovine serum albumin and 97% inhibition of its reaction with the IgG fraction from the immune serum. The tryptic digest was fractionated on Sephadex G50 and the inhibitory activity was recovered completely in the first peak which was further resolved into four components by chromatography on CM-cellulose. The four components each had a molecular weight of about 17 000 and their inhibitory activity ranged between 50 and 80%. The inhibitory activity was destroyed by tryptic digestion at lysine residues or by performic acid oxidation. Therefore, intactness of antigenic reactive sites and conformational integrity due to both size and disulfide bonds are critical for the immunochemical reactivity.

The genetic control of T lymphocyte proliferative response to the five synthetic antigenic sites of myoglobin, two synthetic nonantigenic control peptides, and one "nonsense" peptide was determined in independent and recombinant strains of mice. In all the strains examined, the nonantigenic control peptides and the "nonsense" peptide did not invoke a response in myoglobin-primed mice. Further, when mice were not primed with whole myoglobin, no response was obtained with any of the antigenic sites. Haplotypes H-2d, H-2f, and H-2s are higher responders to sites 1 and 2, whereas haplotypes H-2d and H-2s are high responders to site 5. Response to site 3 may be controlled by a non-H-2-linked gene. Site 4 can stimulate H-2b and H-2k haplotypes that are nonresponders to the whole myoglobin. Studies with the recombinant strains suggested that Ir genes to sites 1 and 2 map in the I-A subregion and I-C subregion and were designated Ir-Mb-1,2(A) and Ir-Mb-1,2(C). Ir genes to sites 4 and 5 mapped only in the I-A subregion and were designated Ir-Mb-4(A) and Ir-Mb-5(A). These studies suggest that individual antigenic sites in a molecule are controlled by unique Ir genes.

Comparisons have been made of the neuraminic acid, hexosamine and hexose content of saliva and bronchial mucus obtained from chronic bronchitis patients and controls. It has been shown that a bronchitic gives a bronchial mucus, which contains on average 30 times more neuraminic acid (per g of non-dialysable material) than is present in his saliva. With controls the neuraminic acid content of their bronchial mucus was on average 8 times greater than that of their saliva. Since the proportion of bronchial mucus in a chronic bronchitic's sputum decreases as he recovers, a patient's progress can be followed chemically by neuraminic acid determination.

RUMEN1 isolated from seal, by chromatography on CM-cellulose in tris buffer at pH 8.5, five myoglobins all with the same iron content and the same N-terminal, but showed differences in isoelectric points. Akeson and Theorell2 isolated, from horse muscle, three myoglobins with similar amino-acid compositions but had charge differences. Sperm whale myoglobin was fractionated by Edmundson and Hirs3, on ‘IRC-50’ into four components, using citrate–chloride buffer at pR 5.86–5.88. However, they encountered zone tailing which, as they pointed out, might have masked the presence of minor components, We have resolved crystalline sperm whale myoglobin into at least twelve components by chromatography. Some properties of these components have been investigated. These included their amino-acid compositions, spectral and electrophoretic behaviour, their molecular weights, and their antigenic character.

Separations of the mucoproteins in a highly viscous sample of chronic bronchitis sputum have been accomplished by electrophoresis on 0.5-mm glass bead columns. At least two proteins were obtained in the homogeneous state; one of these closely resembled serum albumin while the other appeared unique to sputum.

The use of cetyltrimethylammonium bromide as a precipitant of the acidic mucoproteins of chronic bronchitis sputum has been investigated. This reagent together with a cellulose ion exchanger has been used to isolate two electrophoretically homogeneous mucoproteins for which analysis of individual carbohydrates and the nature of the amino acids are reported.

A chemical method for cleavage of C-tryptophyl peptide bonds has been developed and applied to sperm whale myoglobin. The first step involved the oxidation of the tryptophan residues in the protein at 0° and pH 5.0. This was followed by a mild acid hydrolysis step at room temperature. With this procedure 92–94% cleavage was obtained at tryptophan-7 and 17–22% cleavage at tryptophan-14 in sperm whale myoglobin.

1. Oxidation of sperm-whale metmyoglobin and its apoprotein with periodate has been investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH6.8 and 22 degrees consumption of periodate ceased in 3(1/2)hr. at 43 moles of periodate/mole of myoglobin. The two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues were oxidized; serine increased in the hydrolysates from 6 to 9 residues/mol. 3. At pH5.0 and 22 degrees , consumption levelled off in 4(1/2)hr. at 26 moles of periodate/mole of myoglobin and resulted in the modification of the two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues; serine increased from 6 to 7 residues/mol. and, also, ferrihaem suffered considerable oxidation. 4. Oxidation at pH5.0 and 0 degrees resulted at completion (4hr.) in the consumption of 22 moles of periodate/mole of myoglobin and in the modification of the methionine, tyrosine and tryptophan residues. Spectral studies indicated oxidation of the haem group. This derivative reacted very poorly with rabbit antisera to MbX (the major component no. 10 obtained by CM-cellulose chromatography; Atassi, 1964). 5. Oxidation of apomyoglobin at pH5.0 and 0 degrees was complete in 4hr. with the consumption of 7.23 moles of periodate/mole of apoprotein. The rate of oxidation in decreasing order was: methionine; tryptophan; tyrosine; and after 7hr. of reaction the following residues/mol. were oxidized: methionine, 2.0; tryptophan, 1.6; tyrosine, 0.99. No peptide bonds were cleaved. Metmyoglobin prepared from the 7hr.-oxidized apoprotein showed that the reactivity with antisera to MbX had diminished considerably. 6. Milder oxidation of apoprotein (2 molar excess of periodate, pH5.0, 0 degrees , 2hr.) resulted in the modification of 1.66 residues of methionine/mol. Metmyoglobin prepared from this apoprotein was identical with native MbX spectrally, electrophoretically and immunochemically. It was concluded that the methionine residues at positions 55 and 131 were not essential parts of the antigenic sites of metmyoglobin.

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.

Role of the Amino Groups and C-Terminal of Sperm-Whale Myoglobin in the Antigen–Antibody Reaction

1. The reaction of several peptides and proteins with diborane was studied under different conditions to determine those most suitable for the specific reduction of carboxyl groups. 2. In the reaction of model peptides and the cyclic peptides bacitracin and tyrocidin, reduction at 0° was entirely specific for the carboxyl groups without affecting the peptide bonds. Acid amide residues were not reduced. Some tripeptides showed anomalous results in that the C-terminal residue was quite resistant to reduction. 3. Specific reduction of carboxyl groups was achieved in each of the following proteins: human serum albumin, egg albumin, adult human haemoglobin, sperm-whale apomyoglobin, horse heart cytochrome c and egg-white lysozyme. The C-terminal amino acid was usually reduced. 4. Conditions for specific reduction of all available carboxyl groups are not easily found and may vary from one substance to another. Specific reduction of a limited number of available carboxyl groups may be generally accomplished by reactions at −10°. 5. It is suggested that this chemical modification, which has the advantage of permanence, may be useful in studying the role of carboxyl groups in the conformation of proteins and in the biological properties of peptides and proteins.

1. The reactions of beta-propiolactone with amino acids were investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH9.0 and 22 degrees , after 15min. of reaction, at least 85% of each amino acid had reacted, methionine and cystine being the most reactive. 3. At pH7.0 and 22 degrees most amino acids reacted; methionine, cystine and histidine reacted almost entirely, and proline and lysine to a significantly smaller extent. 4. At pH3.0 and 22 degrees further specificity was obtained; methionine and cystine were the only reactive amino acids. 5. Reaction at pH3.0 and 0 degrees was specific for methionine; it was the only amino acid modified even after 145hr. of reaction.

1. The extent of reaction of the protein with 3,3-tetramethyleneglutaric anhydride was measured by determining the loss of lysine and the equivalent appearance of 3,3-tetramethyleneglutarimidolysine in the hydrolysate at various time-intervals. Of the 19 lysine residues in sperm-whale myoglobin, three did not react with 3,3-tetramethyleneglutaric anhydride. Also, the N-terminal valine did not react. 2. Slight spectral changes were observed on modification of myoglobin. 3. Electrophoretic changes were great since the derivative was negative throughout the range pH6–8·8. 4. The derivative did not react with antisera to native sperm-whale myoglobin.

Publisher Summary This chapter deals with the reaction of proteins with citraconic anhydride and the properties of blocked and deblocked citraconyl (CT-) proteins. Other reversible masking reagents are discussed where it is useful to compare the results with such reagents with those obtained with citraconic anhydride on the same protein. Citraconic anhydride (2-methylmaleic anhydride) reacts with amino groups to give two reaction products. Some of the physicochemical and biological properties of deblocked CT-proteins are considered. In certain cases, the corresponding properties of preparations of the same protein after removal of other blocking groups are given, when available, to facilitate direct comparison and to emphasize the merits of citraconic anhydride. The conformation of deblocked CT-myoglobin is examined using optical rotatory dispersion. The deblocked proteins are highly heterogeneous, with partial recovery of enzymatic activity (for lysozyme), immunochemical properties and native conformation. The usefulness of a reversible reagent depends on the ease of removal of the blocking groups under conditions that will not lead to denaturation, on the homogeneity of the preparation on deblocking, and on the extent of recovery of the biological properties and reversion to the native conformation.

Electrophoretically and chromatographically homogeneous myoglobins from human, monkey (Maccacus rhaesus), horse, camel, beef, lamb and goat have been prepared and their conformational and hydrodynamic parameters studied and compared with those of sperm whale myoglobin X. Measurements of Stokes radii and frictional ratios by gel filtration suggested no detectable conformational differences between horse, camel, beef, lamb, goat and sperm whale myoglobins. Human and monkey myoglobins exhibited a slight but definite increase in the Stokes radius and frictional ratio values. Sedimentation studies showed that, except for human and monkey myoglobins, the myoglobins possessed identical sedimentation coefficients, within experimental error at 2.02 ± 0.01 S. The corresponding values of s020,w for human and monkey myoglobins were appreciably lower at 1.85 S and 1.87 S, respectively. Values of f/f0 calculated from s020,w values strongly suggested the presence of less folded or more asymmetric structures in human and monkey myoglobins relative to the others. Spectral studies showed that all the present eight myoglobins had identical spectral properties in the visible and ultraviolet regions. In optical rotatory dispersion (ORD) studies all the present myoglobins gave a negative rotation minimum at 233 nm and a positive extremum at 199 nm. The rotatory behavior of myoglobins from sperm whale, goat, lamb, beef and camel were quantitatively similar, both in [m′]233 nm and in [m′]199 nm. Horse myoglobin showed a slightly lower rotation at the negative minimum at 233 nm and at the positive maximum at 199 nm. Human and monkey myoglobins showed appreciably lower rotations both at 233 nm and at 199 nm. Also the b0 values for human and monkey myoglobins were appreciably lower (−335 and −312, respectively) than the corresponding value for the other myoglobins (−390 to −412).