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Modification of ASI-Casein by 2-phenyl-1,4-dibromoacetoin Beveridge, Herbert James Thomas

Abstract

The histidine specific reagent 2-phenyl-l,4-dibromoacetoin (PDA) has been applied to αSI-casein B. Reaction of αSI-casein with PDA for 26 hours resulted in the loss of about two residues each of histidine and methionine per αSI-casein monomer (molecular weight = 27,000). The modified protein was 90% soluble in 8 mM calcium chloride and precipitated quantitatively at 13 mM calcium chloride. The control was precipitated quantitatively at 8 mM. The calcium binding capacity of the modified αSI-casein was reduced to about 4.5 calcium ions per PDA αsi-casein monomer from 12.4 calcium ions per αSl-casein monomer. Reaction of αSI-casein with PDA resulted in the production of aggregated material which remained at the origin on polyacrylamide gel electrophoresis in the presence of urea and 2-mercaptoethanol and which was eluted at the void volume of a Sephadex G-200 column. While following the time course of the reaction of PDA with αSI-casein, it was found by N-bromosuccinimide (NBS) spectrophotometric titration (6 M urea-acetate-formate buffer, pH 4.0) that two residues of tryptophan per monomer αSI-casein could be detected at zero reaction time but 3.1 residues could be detected after 30 hours. Comparison of the results obtained with αSI-casein, PDA αSl-casein, β-lactoglobulin and α-chymotrypsinogen using both the NBS titration procedure and the p-dimethylaminobenzaldehyde method of Spies and Chambers (46) suggest the presence of a "buried" tryptophan residue in αSI-casein. PDA has a negligible effect on the NBS titration procedure so the residue "exposed" cannot be an artifact generated by PDA bound to the protein. Increasing the urea concentration to 10 M did not expose the third tryptophan residue to NBS.

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