Affinity labelling of liver alcohol dehydrogenase. Effects of pH and buffers on affinity labelling with iodoacetic acid and (R, S)-2-bromo-3-(5-imidazolyl)propionic acid.
Both iodoacetic acid and (R,S)-2-bromo-3-(5-imidazolyl)propionic acid (BrImPpOH) react with liver alcohol dehydrogenase in an affinity labelling mechanism between pH 6.1 and 10.5. The buffer-independent dissociation constants and the first-order rate constants have been determined as a function of pH. With BrImPpOH a pKa close to 9 for the free enzyme is assigned to the zinc-water ionization. The buffers used exerted a protective effect upon the inactivation of the enzyme by iodoacetic acid and BrImPpOH. Phosphate buffer showed a high degree of protection especially at lower pH, while zwitterionic buffers like Mes (4-morpholineethanesulfonic acid), Pipes (1,4-piperazinediethanesulfonic acid), Epps [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid] and Bicine [N,N-bis(2-hydroxyethyl)glycine] gave less protection to various degrees. An exception was Ches (cyclohexylaminoethanesulfonic acid) which had an anomalously high affinity for the iodoacetate binding site. The dissociation constants of the buffers were calculated for the case of inactivation by both iodoacetic acid and BrImPpOH.
D000345:Affinity Labels / Q000494:pharmacology*; D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology*; D007461:Iodoacetates / Q000494:pharmacology*; D019807:Iodoacetic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics
D000345:Affinity Labels; D002021:Buffers; D007093:Imidazoles; D007461:Iodoacetates; C000314:alpha-bromo-beta-(5-imidazolyl)propionic acid; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D019807:Iodoacetic Acid