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M Z Atassi

Publications and source records attributed to M Z Atassi.

At least 217 records · Page 12Linked to original sources
PubMed1977

Antibodies against protein antigenic sites that are identical in the homologous protein of the immunized animal. Autoreactivity in rabbits of antibodies to sperm-whale myoglobin.

Sequence comparisons between the antigenic sites of sperm-whale myoglobin and the corresponding regions in rabbit myoglobin indicate that rabbits make antibodies to regions of the sperm-whale myoglobin molecule which are identical to the corresponding regions in rabbit myoglobin. Rabbit myoglobin did not precipitate with antisera to sperm-whale myoglobin. However, it exhibited an extensive cross-reaction as demonstrated by its ability to inhibit the precipitin reaction of sperm-whale myoglobin, and on an immunoadsorbent, bound a large amount of antibodies to sperm-whale myoglobin.

Amino Acid Sequence↗
PubMed1976

Delineation of the third antigenic site of lysozyme by application of a novel 'surface-simulation' synthetic approach directly linking the conformationally adjacent residues forming the site.

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.

Amino Acid Sequence↗
PubMed1976

A fragment comprising the last third of bovine serum albumin which accounts for almost all the antigenic reactivity of the native protein.

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.

Amino Acid Sequence↗
PubMed1976

Enzymic and immunochemical properties of lysozyme. XVI. A novel synthetic approach to an antigenic reactive site by direct linkage of the relevant conformationally adjacent residues constituting the site.

Previous studies from this laboratory on the immunochemistry of specific chemical derivatives of native lysozyme and of the two disulfide peptide 62-68 (Cys 64-Cys 80) 74-97 (Cys 76-Cys 94) (i.e. (SS)2-peptide), have established an antigenic reactive site to comprise the spatially contiguous surface residues: Trp 72, Lys 97, Lys 96, Asn 93, Thr 89 and Asp 87. In the present work, the identity of the site was verified by an entirely different and novel approach. The aforementioned amino acids were linked directly into a single linear peptide with an intervening spacer where appropriate and substituting phenylalanine for tryptophan (i.e. Phe-Gly-Lys-Asn-Thr-Asp). This peptide (which does not exist in native lysozyme but simulates a surface region of the protein) possessed a remarkable inhibitory activity towards the reaction of lysozyme with its antisera. The immunochemical reactivity of the peptide was equal to the maximum expected reactivity of the site (i.e. a third of the total antigenic reactivity of lysozyme). These findings define quite conclusively and accurately the reactive site which is clearly composed of spatially adjacent residues that are distant in sequence reacting as if in direct linear linkage. The unequivocal establishment of this concept indicates that antigenic sites need not always be composed of residues in direct peptide linkage in the sequence. The nature of the site may depend on the protein. This unorthodox attack at the problem provides a novel and powerful approach for final delineation of the antigenic reactive sites (and perhaps other types of binding sites) in native proteins, following the completion of accurate narrowing down by chemical methods.

Amino Acid Sequence↗
PubMed1976

Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid.

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.

Amino Acids↗
PubMed1976

Enzymic and immunochemical properties of lysozyme. XIII. Accurate delineation of the reactive site around the disulfide 6-127 by immunochemical study of beta-propiolactone lysozyme derivative and of synthetic disulfide peptides.

In previous reports from this laboratory it was shown that an antigenic reactive site resides around the sequences 6-13 and 126-128 linked by the disulfide 6-127. The present work provides a strong support for the location of the reactive site by an independent approach. It also determines accurately the boundaries of the reactive site. 1. The two methionine residues in lysozyme were carboxyethylated by reaction with beta-propiolactone. The electrophoretically homogeneous derivative had no other modified amino acids and showed no conformational changes, relative to native lysozyme, as determined by ORD and CD measurements. However, it exhibited a slight increase in disulfide reducibility relative to native lysozyme and its lytic activity was about half that of native lysozyme, probably as a result of the slight conformational change. On the other hand, the antigenic reactivity of the derivative was equal to that of native lysozyme with several goat and rabbit antisera to lysozyem. It was therefore concluded that methionines 12 and 105 were not parts of antigenic reactive sites in native lysozyme. 2. Eleven peptides, corresponding to various sequences on the two sides of the disulfide 6-127 (i.e. two groups of peptides) were synthesized, purified and characterized. One group (A) of peptides comprised sequences 3-14, 5-14, 6-14, 5-13, 5-12 and an analog of sequence 5-14 in which methionine 12 is replaced by glycine. The second group (B) of peptides comprised sequences 125-129, 125-128, 126-128, 127-128, and 125-127. From groups A and B, nine disulfide-containing peptides (see Fig. 2) were synthesized, purified, characterized and their immunochemical interactions with antisera to native lysozyme studied. Towards each of the antisera studied here, Phe-3, Gly-4, Arg-5, Arg-125 and Leu-129 were not essential parts of the reactive site. On the other hand, Arg-14, Lys-13, Gly-126 and with some antisera Arg-128 were each critical for the reactivity of the site. Peptides from group A alone or group B alone did not inhibit the reaction of lysozyme with its antisera, confirming our previous findings that the integrity of the disulfide bond is essential for bringing the two distant (in sequence) parts of the site together. Finally, replacement of Met-12 by glycine did not influence the immunochemical reactivity of the site, confirming the above conclusion that neither of the two methionine residues takes part in interaction of lysozyme with its antibodies. An accurate delineation of the antigenic reactive site is, therefore derived here and its shape in the three-dimensional structure of native lysozyme is described.

Amino Acid Sequence↗
PubMed1975

Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.

Amino Acids↗
PubMed1975

Enzymic and immunochemical properties of lysozyme. XI. Conformation and immunochemistry of the two-disulfide peptide and the tryptophan and lysine residues in its antigenic reactivity.

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.

Amino Acids↗
PubMed1975

Enzymic and immunochemical properties of lysozyme. IX. Conformation and immunochemistry of derivatives succinylated at certain lysine residues.

Succinylation of lysozyme in the presence of 7 molar excess of [1,4-14C2]-succinic anhydride gave a reaction product which showed at least six components by disc electrophoresis. Chromatography on CM-cellulose enabled the isolation of six homogeneous derivatives. The derivatives were succinylated at the following locations: derivative I, lysines-1 (alpha- and epsilon-NH2), -13, -97 and -116 and the OH group at position 43 (or 36 or 40); derivative II, lysines-1 (alpha- and epsilon-NH2), -13, -96, -116; derivative III, lysines-1 (alpha-and epsilon-NH2), -13, -97, -116; derivative IV, lysines-1 (alpha-NH2), -33, -96 and -116; derivative V, lysines-1 (alpha-NH2), -33 and -96; derivative VI, lysines-33 and -116. Conformational changes were detectable in derivative I by ORD and CD measurements and by accessibility of the disulfide bonds to reduction. On the other hand, the other five succinyl derivatives showed no conformational changes by ORD and CD measurements. However, their disulfide bonds were slightly more accessible to reduction than lysozyme, with the increase being somewhat higher in derivatives I, II and III. Enzymic activity measurements showed that only derivative VI possessed some (10%) enzymic activity. Immunochemical studies with antisera to lysozyme showed that the reactivity of each of the derivatives was lower than the homologous reaction. Correlation of the extent of decrease in immunochemical reaction with the locations of modification and with the results of conformational analysis, led to the conclusion that lysines 33, 96 and 116 are part of antigenic reactive regions in lysozyme. The modification results are also discussed in relation to the three-dimensional structure of lysozyme in solution.

Anhydrides↗
PubMed1975

2,3-Dioxo-5-indolinesulfonic acid, a new highly specific reagent for modification of tryptophan in peptides and proteins.

A new reagent, 2,3-dioxo-5-indolinesulfonic acid (DISA), has been investigated for its specificity to react with tryptophan and tryptophan residues in peptides and proteins. On reaction of 19 amino acids with 1 M excess of DISA in 0.1 M acetic acid (pH 2.9), considerable (50%) modification of tryptophan was obtained within 50 min and no other amino acid was modified. After reaction for 5 h, only proline showed very slight (7%) modification. On reaction in the presence of 5 M excess of DISA, tryptophan was very rapidly modified. Modification of proline became appreciable in the presence of this molar excess of reagent and cysteine modification, although much smaller, became detectable (12 h, 7%). However, proline modification was completely prevented when this amino acid was engaged in a peptide linkage, even after reaction for 45 h in the presence of 5 or 10 M excess of DISA per proline residue. Reaction of egg albumin with 50 M excess of DISA was entirely specific for tryptophan and showed no modification of proline or cysteine residues. The reagent offers the advantages of stability, easy handling, high water solubility and high specificity. It affords protein and peptide derivatives that are completely water soluble because of the polar nature of the added group. The yellow color (lambda max, 367 nm) of the derivatives offers advantages of easy determination of the extent and location of the modification.

Amino Acids↗
PubMed1975

Conformational studies on modified proteins and peptides. IX. Conformation and immunochemistry of hemoglobin reduced at some carboxyl groups by diborane.

A derivative of apohemoglobin was prepared by reduction with diborane at -10 degrees C for 2 h. Four aspartate and two glutamate side chains were reduced per half molecule to their corresponding alcohols. The modifications were identified as: alpha asp 64, alpha-asp 74, alpha-asp 75 and alpha asp 85;beta-glu 121 and beta-glu 6 (or beta-glu 7). Recombination of reduced apohemoglobin with unmodified ferriheme yielded the corresponding reduced hemoglobin derivative which was electrophoretically homogeneous. Hemoglobin and reduced hemoglobin had different electrophoretic mobilities and showed slight spectral differences. Titration with ferriheme revealed that reduced globin possessed an anomalous heme-binding behavior. The s 20,w of reduced hemoglobin was slightly higher than that of hemoglobin. Agregation of the derivative was also apparent from its behavior in gel filtration. ORD and CD measurements indicated substantial conformational differences between hemoglobin and its derivative. Immunochemical studies showed that the derivative reacted poorly with antisera to hemoglobin and, conversely, hemoglobin showed little reaction with antisera to the derivative. The results are discussed in terms of the three-dimensional structure of hemoglobin in the crystalline state and its relationship to the structure in solution.

Amino Acids↗