Abstract
Abstract Harbor seal (Phoca vitulina) and sperm whale (Physeter catodon) myoglobins were carboxymethylated in 0.2 m bromoacetate at pH 6.8. The modification of histidine residues leveled off after approximately 6 and 8 days, respectively, of reaction at 25° in the dark. The NH2-terminal glycine and valine residues, respectively, were modified, as were 1 or 2 lysine residues on the average. The modified seal protein was subjected to digestion with trypsin and the acid-insoluble fraction from that treatment further exposed to thermolysin. The modified sperm whale protein was subjected to thermolysin alone. Peptides containing histidine or a derivative were isolated and identified, and conclusions concerning the patterns of reactivity were tabulated. The results with the sperm whale protein confirm and complete the assignments reported previously (Hugli, T. E., and Gurd, F. R. N. (1970) J. Biol. Chem. 245, 1939–1946). Histidine residue 64 was found to be unreactive, while the peculiar suppression of reactivity of residue 36 relative to the reaction product obtained in the crystalline state was confirmed. The pattern of reactivity of the harbor seal protein was similar. The freely reactive histidine residues were 8, 81, 113, and 116, the unreactive residues were 24, 64, 82, 93, and 97, and those showing various patterns of restriction were 36, 48, 119, and 152.
Highlights
Xaferials-The main fractions of harbor seal (Phoca vitulina) and sperm whale (Plzyseter c&o&n) myoglobins were prepared by the methods of Hapner et al [13]
For the sperm whale protein the appropriate treatment period was chosen as 8 days
Isolation of Sperm TT’haZe Xyoglobin Peptides Containing Ilistidine Residues-The findings reported above on the carboxymethylation pattern of the harbor seal histidine residues are in some respects more clear-cut than those reported previously by
Summary
Xaferials-The main fractions of harbor seal (Phoca vitulina) and sperm whale (Plzyseter c&o&n) myoglobins were prepared by the methods of Hapner et al [13]. Protein preparations were deionized when necessary to the point where pH values matched those previously determined [13]. Polyacrylamide gel electrophoresis (2, 151, PI-I-stat procedures (a), amino acid analyses (2, 17, al), spectrophotometric procedures [13], and pH measurements [14] were performed as previously described. Reaction with Bromoacetafe-The alkylation of harbor seal myoglobin with bromoacetate was carried out with a 2% deionized protein solution and a final concentration of bromoacetate of 0.2 M, adjusted to pH 6.8 before addition to the protein. The column was developed with a series of gradients at 60 ml per hour as described in the text. The effluent was continuously monitored following alkaline hydrolysis and ninhydrin color development.
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