Intramolecular Disulphide Bridges Research Articles

The nature of the heterogeneity of prothrombin during dicoumarol therapy.

THE effect of the dicoumarol drugs on clotting factor biosynthesis is similar to that of vitamin K deficiency, in that the coagulant activities of clotting Factors II, VII, IX and X in the plasma are reduced. Also, levels are rapidly restored by the administration of vitamin K. The burst of clotting factor synthesis immediately following vitamin K treatment can take place even in the presence of antibiotics which inhibit general protein synthesis in the liver1. This suggests that vitamin K may act at a stage after peptide chain completion, such as the formation of intramolecular disulphide bridges, attachment of carbohydrate or secretion of the glycoprotein into the plasma. If antagonists of vitamin K interfere with the completion of the clotting proteins, incomplete forms may be present in the plasma during dicoumarol therapy and the nature of these forms may give a clue to the action of vitamin K. To test this hypothesis we have isolated the prothrombin of patients receiving dicoumarol therapy and investigated some of its properties.

The effect of cross-links on the mobility of proteins in dodecyl sulphate-polyacrylamide gels.

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.

Reaction of the sulphydryl groups of lobster-muscle glyceraldehyde-3-phosphate dehydrogenase with organic mercurials

1. 1.|Crystalline lobster-muscle glyceraldehyde-3-posphate dehydrogenase ( d-glyceraldehyde-3-phosphate: NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) possesses 7.3 ± 0.3 cysteine residues per mole of enzyme (140 000 g), i.e. approx. 2 cysteine residues per polypeptide chain, which are extremely reactive toward organic mercurials; 8 of the 20 cysteine residues of the enzyme are totally unreactive toward organic mercurials. 2. 2.|Enzyme carboxymethylated at each of the 4 ‘active site’ cysteine residues possesses 7.4 ± 0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate and cannot be distinguished from native enzyme on the basis of these titrations. 3. 3.|Enzyme treated with iodosobenzoic acid and containing an intramolecular disulphide bridge at the ‘active center’ possesses 4.0 ± 0.3 sulphydryl groups which are extremely reactive toward p-mercuribenzoate, i.e. approx. 4 less than native enzyme. 4. 4.|Under normal conditions, lobster-muscle glyceraldehyde-3-phosphate dehydrogenase is 90–95% inactivated concomitant with the formation of 8 moles of mercaptide per mole of enzyme (140 000 g); inhibition takes place in a linear manner with respect to mercurial concentration. 5. 5.|In the presence of a large molar excess of NAD +, the formation of 4 moles of mercaptide per mole of enzyme (140 000 g) has no inhibitory effect and must be attributed to reaction of a ‘nonessential’ sulphydryl group.