Interactions of Alcohol Dehydrogenase to p‐Hydroxyacetophenone–sepharose and p‐Acetamidophenol‐sepharose

Abstract

p-Hydroxyacetophenone (HAP)-sepharose is known to be an effective ligand for isolation of aldehyde dehydrogenase and chloramphenicol acetyltransferase. In this study, we investigated ligand specificities to alcohol dehydrogenase (ADH) using p-HAP-sepharose and p-acetamidophenol (AAP)-sepharose. p-HAP and p-AAP were coupled to epoxy-activated sepharose and used as ligands for affinity chromatography to detect binding proteins. Fractions of affinity chromatography were collected and separated by SDS-PAGE. Commassie brilliant blue staining was carried out for detecting proteins. For determining protein sequences, polypeptide was separated by HPLC after cleavage of Lys-specific protease digestion. p-HAP ligands have the ability to bind to seven proteins, including class I ADH extracted from a rat cytosolic fraction as well as HLADH (horse liver ADH). p-HAP, p-AAP and benzaldehyde (10 mM each) could elute HLADH from the complex of HLADH-pHAP-sepharose column. On the other hand, p-AAP-sepharose has no ability to bind to HLADH, although three proteins from a rat liver cytosolic fraction were found to be able to bind to p-AAP-sepharose. p-HAP binds to ADH in a structure-specific manner and this binding is nonspecific to species of origin of ADH.