Synthesis and use of bifunctional chloromethylalkanedione derivatives of variable chain length for cross-linking thiol groups in oligomeric proteins. Specific cross-linking in glyceraldehyde 3-phosphate dehydrogenase.

Bischloromethylpentanedione, bischloromethylhexanedione, bischloromethyloctanedione and bischloromethyldecanedione were synthesized from their corresponding dicarboxylic acids via the bis-acyl chloride and the bisdiazomethylketone derivatives. These compounds proved to be highly specific cross-linking reagents for rabbit skeletal-muscle glyceraldehyde 3-phosphate dehydrogenase. Incubation of the enzyme with cross-linking reagents resulted in both a time- and concentration-dependent formation of covalently linked oligomeric structures. The major cross-linked product detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was the dimer (mol. wt. 72000). Sepharose 6B chromatography of the cross-linked enzyme showed that it still existed as the tetramer. Cross-linking was dependent on the native structure of the enzyme, since it was abolished on denaturation of the enzyme. The actual covalently linked product depends on the conditions of modification and the chain length of the reagent. The maximum yield of dimer (70-80%) was obtained with bischloromethylhexanedione, and the yield decreased with either shorter- or longer-chain compounds. The calculated distance between the two reactive points in bischloromethylhexanedione is 1.21-1.45nm. Bischloromethylhexanedione modified at least two thiol groups per monomer. Modification of the active-site thiol, cysteine-149, was not essential for cross-linking, since glyceraldehyde 3-phosphate dehydrogenase carboxymethylated on cysteine-149 still reacted to form the dimer. The rate of chemical cross-linking was markedly decreased by increasing the NAD(+) occupancy of the enzyme active sites. These experiments are discussed in terms of the asymmetry of the enzyme structure in solution.