High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase.
Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004800:Enzymes, Immobilized; D006736:Horses; D008099:Liver / Q000201:enzymology; D011485:Protein Binding; D012822:Silicon Dioxide
D004800:Enzymes, Immobilized; D012822:Silicon Dioxide; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase