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The interferences of bilirubin and hemoglobin were tested in two cholesterol procedures in which enzymes were used as chemical reagents. Both procedures used similar approaches with cholesterol esterase to free cholesterol from its esters and cholesterol oxidase to generate hydrogen peroxide from the total free cholesterol resulting. From that common start, one procedure then used catalase to generate formaldehyde from methanol and the peroxide produced from cholesterol, and the formaldehyde was then reacted with acetylacetone to produce a yellow chromogen, while the other procedure used peroxidase to catalyze a reaction directly between peroxide and 4-aminoantipyrine plus phenol to generate a pink chromogen. Bilirubin and hemoglobin were shown to produce some interference by reacting competitively with peroxide in both systems and by contributing residual absorbance at the wavelengths of measurement of each of the chromogens. Since bilirubin showed a spectral change, static blanking with sample blanks caused overcorrections. However, the elimination of a sample blank for either procedure could result in a favorable compensating error because the residual color of bilirubin could substitute in part at least for the lost reactivity of the peroxide used up in reaction with the bilirubin of the sample.

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.

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.

Previous work from this laboratory on the antigenic structure of myoglobin (Mb) has shown that the antigenic reactivity of peptide 1–55 resides entirely in the shorter peptide 1–31 and is located within sequence 13–23. Narrowing down this region even further was achieved here by studying chemical derivatives of peptide 1–31. Five derivatives of this peptide were prepared, characterized and their immunochemistry and conformation studied. Modification of tryptophan residues 7 and 14 with dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide resulted in retention of complete antigenic reactivity. In contrast, succinylation of lysine 16, or reduction of glutamic acid 18 and aspartic acid 20 (or 27) by diborane obliterated antigenic reactivity entirely. Also, a derivative carboxymethylated at histidines 12 and 24, which has been shown to retain full antigenic reactivity, lost this reactivity completely when it was succinylated at lysine 16. Reaction of peptide 1–31 with diazonium-1H-tetrazole gave rise to a derivative modified at lysine 16, histidines 12 and 24 and tryptophan residues 7 and 14. This derivative had no ability to react with antisera to myoglobin. Since none of the derivatives exhibited any changes in conformation, these results indicated that tryptophans 7 and 14 are located outside the reactive region. On the other hand, lysine 16, glutamic acid 18 and possibly aspartic acid 20 are located within the antigenic reactive site. This information, together with our previously reported studies, has shown that the first third of the myoglobin molecule (i.e. sequence 1–55) has only one antigenic reactive region and it is located within (but does not necessarily include all of) sequence 15–23. Glycine 23 is probably unessential for its reactivity. From the present findings, and the overall strategy of approach, it was concluded that, at this level of coarse delineation (i.e. down to 8–9 residues), reactive regions are surprisingly sharp but may be more diffuse in unfolded proteins. A reactive region is invariably so but its efficiency varies with the serum. When a reactive region is segmented, its two portions may react but with decreased efficiency. Therefore, before fragmentation of a protein for immunochemical studies, it is best to confirm that the location of scission is outside a reactive region. Other precautionary steps and critical analysis of approaches employed in studying the antigenic structures of proteins are discussed.