Analysis of the reconstitution of oligomeric enzymes by crosslinking with glutaraldehyde: kinetics of reassociation of lactic dehydrogenase

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Abstract
Cross-linking with glutaraldehyde with subsequent NaDodSO4[sodium dodecylsulfate]-polyacrylamide gel electrophoresis was introduced as a convenient method for studying the association of oligomeric proteins (Hermann et al, 1979). An improved version of this approach was applied to the analysis of the complex association behavior of the tetrameric lactic dehydrogenase from pig muscle. Monomers, dimers (as intermediates of reconstitution) and tetramers could be quantitatively determined during reconstitution. The initial fast formation of dimers from monomers does not reach completion; a certain amount of monomers remains during the whole reconstitution process. Monomers and dimers disappear parallel to the formation of tetramers. The reassociation behavior of lactic dehydrogenase is described by a kinetic mechanism comprising a dissociation-association equilibrium of mnomers and dimers [characterized by an equilibrium constant K = (3 .+-. 1) .times. 108 l mol-1] followed by the rate-limiting association of dimers to tetramers [described by a 2nd-order rate constant k = (3.15 .+-. 0.15) .times. 104 l mol-1 s-1]. Tetramerization strictly parallels reactivation.