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Chemically Crosslinked Lactate Dehydrogenase: Stability and Reconstitution After Glutaraldehyde Fixation
Author(s) -
Gottschalk N.,
Jaenicke R.
Publication year - 1987
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1987.tb00486.x
Subject(s) - cooperativity , chemistry , denaturation (fissile materials) , glutaraldehyde , protein quaternary structure , guanidine , enzyme , kinetics , lactate dehydrogenase , guanidinium chloride , reaction rate constant , thermal stability , dehydrogenase , native state , crystallography , stereochemistry , biochemistry , chromatography , nuclear chemistry , protein subunit , organic chemistry , physics , quantum mechanics , gene
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 × 10(‐4) and 2.7 × 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.