The effect of cryosolvents on the spectral and catalytic properties of liver alcohol dehydrogenase.
The effects of aqueous organic cryosolvents on the structural and catalytic properties of horse liver alcohol dehydrogenase (liver alcohol dehydrogenase) have been investigated. The cosolvents studied were ethanol, methanol, dimethyl sulfoxide, and dimethylformamide. All show potential as cosolvents for cryoenzymological investigations of the catalytic action of liver alcohol dehydrogenase. Limitations due to the formation of abortive complexes, or the cosolvent acting as a substrate were considered. Possible adverse structural effects of the cosolvents were ascertained by utilizing the intrinsic fluorescent properties of the enzyme. Catalytic effects, as inferred from steady state kinetic studies, were determined from both the oxidation of ethanol and the reduction of p-nitroso-N,N-dimethylaniline, a chromophoric aldehyde analog. It is concluded that each of these solvent systems may be useful for studying certain aspects of the liver alcohol dehydrogenase catalytic mechanism at subzero temperatures. Thus, although the formation of ternary enzyme-cosolvent complexes may restrict the use of cosolvents in some experiments, no apparent adverse effects are observed on the enzyme structure, coenzyme binding, or catalytic reactions. A number of interesting features were observed. For example, fluorescence titration of the native enzyme near 0 degrees C in either aqueous solution or 50% dimethyl sulfoxide revealed pK values in the vicinity of 10.5 and 12.5, in contrast to the previously reported single pK of 9.2 observed in aqueous solution at 25 degrees C.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004126:Dimethylformamide / Q000494:pharmacology*; D000431:Ethanol / Q000494:pharmacology*; D005615:Freezing; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D000432:Methanol / Q000494:pharmacology*; D010084:Oxidation-Reduction; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D012997:Solvents; D000431:Ethanol; D004126:Dimethylformamide; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D000432:Methanol; D004121:Dimethyl Sulfoxide