Peptides containing the four cysteine and two half-cystine residues labelled with [2-14C]iodoacetic acid were isolated from thermolytic digests of reduced and S-carboxymethylated ovalbumin by paper ionophoresis, pH 6·4, descending paper chromatography and another ionophoresis at pH 1·9. These peptides were analysed for amino acids and the peptides identified with their position in the known linear sequence of the molecule.The location of the disulfide bond for three ovalbumins of differing heat stability were determined by blocking the cysteine residues with non-radioactive iodoacetic acid, reducing the disulfide bond and labelling the half-cystine residues with [2-14C]iodoacetic acid. Radioactive peptides were isolated after thermolytic digestion by paper ionophoresis and descending paper chromatography. The amount of radioactivity on each peptide was quantitated and the two half-cystine labelled peptides accounted for over 90% of radioactivity.The disulfide bond was found to involve the sequences:Phe-Gly-Asp-SerP-De-Glu-A1a-Gln-Cys-Gly-Thr-Ser and Leu-Gln-Cys for the three ovalbumins of differing heat stability. Therefore thiol-disulfide interchange is not involved in the formation of these ovalbumins.

Ovalbumin isolated from eggs of the Japanese quail, C. c. japonica, was subjected to limited proteolysis by subtilisin to give plakalbumin and then fractionated on Sephadex G75 in acid-urea to give plakalbumin S-protein and S-peptide. The plakalbumin peptide was recovered, oxidized with performic acid, and the sequence of amino acids determined from the peptides formed by enzyme digestion. There were two cysteine residues in the 33-residue sequence. The ovalbumin was also oxidized with performic acid and digested with thermo lysin and pepsin before isolating, from a sulfonated polystyrene column, the acidic cysteic acid peptides, as well as acetylated N-terminal peptides and phosphorylated peptides, and determining their amino acid sequence. Additional peptide sequences containing cysteine or half-cystine were characterized.Quail ovalbumin was reduced and carboxymethylated with [2-'4C]iodoacetic acid. Peptides containing labelled S-carboxymethy1cysteine residues were isolated from thermolytic digests of the carboxymethylated ovalbumin by paper ionophoresis and chromatography. Their amino acid sequence was determined and five different sequences involving labelled S-carboxymethy1cysteine residues were established.The presence of two half-cystine residues and the location of the disulfide bond were shown by blocking the cysteine residues with non-radioactive iodoacetic acid, reducing the disulfide bond and labelling the half-cystine residues with [2-'4C]iodoacetic acid. After thermolytic digestion of the protein, radioactive peptides were isolated by paper ionophoresis and chromatography. These studies have thus shown that quail ovalbumin contains one cystine residue and three cysteine residues, which is one residue of cysteine less than in ovalbumin from the hen (Gallus gallus domesticus). There is strong homology in the amino acid sequences of hen ovalbumin and quail ovalbumin determined in these investigations.

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds.Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1 % (w/v) sodium dodecyl sulfate at pH 8·2 with 5,5' -dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.

No abstract