M.Z. Atassi

Immunochemistry of sperm-whale myoglobin: XVIII. Accurate delineation of the single reactive region in sequence 120–153 by study of synthetic peptides+

Abstract Previous reports from this laboratory have shown that in the entire sequence 120–153 of myoglobin (Mb), a single antigenic reactive region existed within sequence 140–151. In the preceding communication, it was demonstrated that lysines 140 and 145 were not essential parts of a reactive region in native Mb. It was therefore concluded that the reactive region occupied the sequence 146–151. In the present paper, strong support from an independent approach is provided by studying the immunochemistry of synthetic peptides corresponding to various parts of the region. Peptides 146–151, 145–151, 146–153 and 147–153 were prepared by solid phase peptide synthesis, purified, characterized and their interaction with three different antisera to Mb studied. With antiserum G1, peptides 145–151, 146–151 and 146–153 possessed comparable inhibitory activities of the reaction of Mb with its antisera. For this antiserum, therefore, residues 145 on one side and 152 and 153 on the other added no contribution to the inhibitory ability, and the reactive region against this antiserum occupied sequence 146–151 entirely. Similarly, in antiserum G3, specificity was directed against a reactive region occupying sequence 146–151. With antiserum G4, the inhibitory abilities of peptides 146–151 and 146–153 were comparable but appreciably lower than that of peptide 145–151. For antiserum G4, lysine 145 may be part of the antigenic reactive region of the free peptide. With the present antisera, peptide 147–153 exhibited a negligible inhibitory activity. From these findings, it was concluded that the antigenic reactive region in segment 120–153 occupies sequence 146–151 invariably, + lysine 145 with some antisera.

Immunochemistry of sperm whale myoglobin VI. Preparation and conformational analysis of eight mammalian myoglobins

Abstract 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).

Immunochemistry of sperm-whale myoglobin. XIX. Accurate delineation of the single reactive region in sequence 54-85 by immunochemical study of synthetic peptides.

Abstract In previous reports from this laboratory it was shown that a single antigenic reactive region existed within the sequence 54–85 of myoglobin (Mb). The reactive region was most likely located around sequence 56–63. In the present communication, strong support for the location of the reactive region is provided from an independent approach by studying the immunochemistry of synthetic peptides corresponding to various parts of the region. Five peptides corresponding to sequences 56–62, 54–62, 57–63, 56–63 and 54–63 were prepared by solid-phase peptide synthesis, purified, characterized and their immunochemical interaction with four different antisera to Mb studied. Towards each of the antisera studied here, Glu-54, Met-55 and Lys-63 were not essential parts of the reactive region. On the other hand deletion of Lys-56 without change in size of peptide (by addition of Lys-63, i.e. peptide 57–63) impairs the immunochemical reactivity of the peptide appreciably. The results, therefore showed that, for each of the antisera studied, the reactive region occupied precisely sequence 56–62. This is a small (seven residues) highly accessible surface location on the corner between helices D and E of Mb.

Enzymic and immunochemical properties of lysozyme: VII. Location of all the antigenic reactive regions. A new approach to study immunochemistry of tight proteins

Abstract 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.

Immunochemistry of sperm-whale myoglobin: XVII. Conformation and immunochemistry of derivatives modified at lysines 98, 140 and 145 by reaction with 3,3-tetramethyleneglutaric anhydride

Abstract 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.

Immunochemistry of sperm whale myoglobin VII. Correlation of immunochemical cross-reaction of eight myoglobins with structural similarity and its dependence on conformation

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.

Conformational studies on modified proteins and peptides. VII. Conformation of ε-prototoxin and ε-toxin from Clostridium perfringens. Conformational changes associated with toxicity

Abstract 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.

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.

Desulfurization of sulfur amino acids and proteins with Raney nickel.

Abstract 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.

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

Abstract 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.