Carboxymethylation of Sperm Whale Myoglobin in the Crystalline State

Crystals of sperm whale ferrimyoglobin were treated with 0.2 m sodium bromoacetate in concentrated ammonium sulfate solution at pH 6.8 for 10 days. The product obtained after dissolving the crystals retained most of the physical properties of the unmodified protein, except for the development of a small number of electrophoretic components representing an increment of about six to eight net negative charges on the average, judged at pH 9.2. The most fully studied preparation contained 6.4, 2.7, 2.0, and 0.8 residue per molecule of histidine, 1,3-dicarboxymethylhistidine, 3-carboxymethylhistidine, and 1-carboxymethylhistidine, respectively. The amino-terminal valyl residue was largely alkylated. Between 1 and 2 residues of lysine were alkylated. No modification of methionine was detected. After removal of heme the modified protein was subjected to cleavage by trypsin and chymotrypsin and the peptides were isolated by chromatography on ion exchange resins and, as required, by paper chromatography or paper electrophoresis. The state of alkylation of 10 of the 12 histidyl residues was established by quantitative estimation of the yields of appropriate tryptic and chymotryptic peptides. The state of residue 12 was determined qualitatively, and that of residue 113 surmised by difference. The following residues were recovered as unmodified histidine: 24, 48, 82, 93, and 97. Residue 119 was recovered primarily in the unmodified form but partly as 1-carboxymethylhistidine. The following residues were recovered primarily or exclusively as 1,3-dicarboxymethylhistidine: 12, 81, 114, and 116. Residue 116 was recovered partly as 3-carboxymethylhistidine. Residue 36 was recovered in high yield exclusively as 3-carboxymethylhistidine. Without exception these results appear to conform to the implications of the crystalline structure reported by Kendrew and Watson when account is taken of the geometry of interactions between neighboring myoglobin molecules in the crystal lattice.