The enzyme, yeast alcohol dehydrogenase, was examined by differential scanning calorimetry and fluorescence spectroscopy. As indicated by the magnitude of the temperature at which the heat effect due to unfolding is maximal, and the temperature range over which unfolding occurs, the yeast enzyme appears to be less stable than its horse liver counterpart. Additionally, altering the pH from 6.0 appears destabilize the enzyme. The spectral shift in maximal emission wavelength that accompanies an alteration in pH suggests that structural changes occur at extreme values of acid and alkaline pH. When slow scan rate experiments were performed, two separate heat effects could be resolved. The results of our and prior fractionation work indicate that the two heat effects are likely due to two molecular species, and not domains within the YADH molecule.
The enzyme, yeast alcohol dehydrogenase, was adsorbed to porous nitrocellulose and nylon membranes. The two membranes provide different surface chemistries as indicated by the results of the streaming potential, enzyme adsorption, and fluorescein isothiocyanate adsorption experiments. The stability of the enzyme, as determined by continually measuring the extent of coenzyme reduction as a function of time, appeared to be much less for the enzyme adsorbed to the positively charged membrane surface. Moreover, the enzyme adsorbed to the positively charged membrane was the least responsive to pulses of the reducing agent, dithiothreitol, and appeared to exhibit the highest transition temperature when subjected to differential scanning calorimetry analysis. These results indicate that the entropically spreading process observed for other adsorbed proteins may be occurring and the process is more rapid and extensive when enzyme is adsorbed to the nylon than the nitrocellulose membrane. In addition to the relative stability of the enzyme on two different surfaces being examined, the effect of the microenvironment on modulating the activity of the enzyme was investigated by using the reversibility of the enzyme-catalyzed reaction as a probe of the average local environment of the enzyme. It was found that a threshold buffer concentration existed that, once exceeded, the effect of proton production by the reaction could be suppressed.
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