Coupling reaction of lactate dehydrogenase after reversible masking of the SH-groups. Reaction with p-diazobenzenesulfonic acid for marking essential histidine or tyrosine residues

Abstract

For some time there have been indications of the importance of histidine and tyrosine for the catalytic activity of lactate dehydrogenase. The presence of reactive sulfhydryl groups in this enzyme has made it difficult to modify the histidine and tyrosine groups chemically without destroying the SH‐groups at the same time. We have thus attempted to mask all the sulfhydryl groups reversibly so that the reaction of diazotised [35S]sulfanilic acid with the protein could be examined.The essential SH‐groups of the protein could be protected by adding a small excess of p‐chloromercuribenzoate or by forming a mixed disulphide with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). The organo‐mercury derivative of lactate dehydrogenase, which had a residual activity of 13%, recovered 60% of the original enzymic activity on standing in pyrophosphate buffer, pH 8.5. The enzyme could not be fully reactivated on adding glutathione. Under the same conditions, there was a slow liberation of 2‐nitro‐5‐mercaptobenzoate from the enzyme which had reacted with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). Coupling these modified lactate dehydrogenase preparations resulted in a fall in the SH‐titre of the enzyme. An attempt was thus made to mask all the sulfhydryl groups of the enzyme reversibly by means of the above mentioned reagents. When more than 1.5 g molecules of SH‐groups/36,000 g protein had reacted it was no longer possible to achieve 100% reactivation with cysteine.In a final attempt, the complex of lactate dehydrogenase with mercuric chloride was prepared. This derivative was inactive and contained no free thiol groups. Both the original enzymic activity and SH‐titre were fully restored with cysteine. Like the native enzyme, Hg‐lactate dehydrogenase also binds 4 molecules of the NAD‐sulphite complex, albeit with an increased dissociation constant.Hg‐lactate dehydrogenase was coupled with diazotised [35S]sulfanilic acid. The reaction was stopped with resorcinol and the reaction mixture was treated with cysteine. By this procedure the enzyme could be inhibited without loss of SH‐groups. The specific enzymic activity of the inhibited enzyme was proportional to the incorporation of 35S up to a value of 1.5 azoresidues/subunit. Azo‐lactate dehydrogenase migrated to the anode more rapidly than the native enzyme during electrophoresis at pH 8.5.