Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus

Open Access
Publisher Website
Google Scholar
Abstract
Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 ± 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine · HCl concentrations is characterized by a two-step profile: at 0.4–0.8 M, partial unfolding is parallelled by inactivation; at 2.0–2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4–1.8 M guanidine · HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine · HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine · HCl, as monitored by the decrease of protein fluorescence, is fast (Colloq. Ges. Biol. Chem. Mosbach 34, 62–90].

This publication has 36 references indexed in Scilit: