Chemically Crosslinked Lactate Dehydrogenase: Stability and Reconstitution After Glutaraldehyde Fixation

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Abstract
Lactate dehydrogenase (LDH) from pig heart was stabilized in its tetrameric state by crosslinking with glutaraldehyde. The product (containing 82% tetramers, 9% dimers, 6% monomers, and 3% aggregates) retained up to .apprxeq.60% of its original activity without detectable changes in the spectral and catalytic properties of the enzyme. Fixation of the native quaternary structure enhances the stability of the enzyme: the equilibrium transitions of heat and guanidine deactivation are shifted from 60 to 65.degree. C and cG.times.HCl = 0.30 to 1.0 M, respectively. Deactivation and denaturation of the crosslinked enzyme do not coincide: the equilibrium transition of fluorescence emission at cG.times.HCl .apprxeq. 3.5 M is paralleled by a significant decrease in cooperativity. The kinetics of thermal denaturation allow structural transitions within a partially crosslinked, native-like species (.ltoreq.10%) to be separated from the unfolding of the fully crosslinked enzyme. The corresponding first-order rate constants at cLDH = 10 .mu.g/ml and 60.degree. C are 4.5 .times. 10-4 and 2.7 .times. 10-5 s-1, respectively. The yield of reconstitution of the crosslinked enzyme after acid denaturation does not exceed 30%; the rate of reactivation is fast due to the significant residual structure of the acid-denatured enzyme. Rate-determining association (which dominates the reconstitution of the unmodified enzyme) does not contribute to the kinetic mechanism. After complete randomization in 6 M guanidinium chloride, incorrect near-neighbor interactions between the crosslinked subunits essentially block the reconstitution of the native structure and function.