Yeast alcohol dehydrogenase bound to membranes: surface and microenvironment effects on activity and stability.
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.
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
C L
CL
Kennedy
M M
MM
Domach
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D002021:Buffers; D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002621:Chemistry; D003101:Collodion / Q000378:metabolism; D004229:Dithiothreitol; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D009243:NAD / Q000378:metabolism; D009757:Nylons / Q000378:metabolism; D010084:Oxidation-Reduction; D010710:Phosphates; D011522:Protons; D013696:Temperature; D013861:Thiocyanates; D013997:Time Factors; D015003:Yeasts / Q000201:enzymology*
D002021:Buffers; D004800:Enzymes, Immobilized; D005452:Fluoresceins; D008567:Membranes, Artificial; D009757:Nylons; D010710:Phosphates; D011522:Protons; D013861:Thiocyanates; D009243:NAD; D003101:Collodion; D000426:Alcohol Dehydrogenase; D016650:Fluorescein-5-isothiocyanate; D004229:Dithiothreitol