Chemically crosslinked lactate dehydrogenase: stability and reconstitution after glutaraldehyde fixation.
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 approximately equal to 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 degrees C and CG.HCl = 0.3 to 1.0 M, respectively. Deactivation and denaturation of the crosslinked enzyme do not coincide: the equilibrium transition of fluorescence emission at CG.HCl approximately equal to 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 (less than or equal to 10%) to be separated from the unfolding of the fully crosslinked enzyme. The corresponding first-order rate constants at CLDH = 10 micrograms/ml and 60 degrees C are 4.5 X 10(-4) and 2.7 X 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.
D000818:Animals; D003432:Cross-Linking Reagents; D004795:Enzyme Stability; D005976:Glutaral; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D013552:Swine; D013816:Thermodynamics
D003432:Cross-Linking Reagents; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D005976:Glutaral