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

Abstract

Bischloromethylpentanedione, bischloromethylhexanedione, bischloromethyloctanedione and bischloromethyldecanedione were synthesized from their corresponding dicarboxylic acids via the bis-acyl chloride and bisdiazomethylketone derivatives. These compounds were highly specific cross-linking reagents for rabbit skeletal-muscle glyceraldehyde 3-phosphate dehydrogenase [EC 1.2.1.12]. Incubation of the enzyme with cross-linking reagents resulted in a time- and concentration-dependent formation of covalently linked oligomeric structures. The major cross-linked product detected by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis was the dimer (MW 72,000). 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 depended on conditions of modification and chain length of the reagent. The maximum yield of dimer (70-80%) was obtained with bischloromethylhexanedione, and the yield decreased with shorter- or longer-chain compounds. The calculated distance between the 2 reactive points in bischloromethylhexanedione was 1.21-1.45 nm. Bischloromethylhexanedione modified at least 2 thiol groups/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 were discussed in terms of asymmetry of the enzyme structure in solution.