HIGH-RESOLUTION OF MULTIPLE FORMS OF RABBIT RETICULOCYTE HEXOKINASE TYPE-I BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY

Hydrophobic interaction chromatography (HIC) has been employed extensi vely in the separation of proteins by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this study, a new hydrophobic interaction chromatographic support, Toyopearl Phenyl 650 S, was investigated in order to examine the dist ribution of multiple forms of rabbit reticulocyte hexokinase type I. T hese distinct forms of the enzyme, designated hexokinase Ia, Ia and I b, show similar kinetic and physical properties, similar molecular mas ses (ca. 100 000) and a different intracellular distribution. The resu lts obtained using Toyopearl Phenyl 650 S of 20-50-mu m particle diame ter show that this HIC support allows very high resolution, comparable to that obtainable with HIC-HPLC columns but with the advantage of ch arging a higher amount of starting material even with a high protein c oncentration. These characteristics render Toyopearl Phenyl 650 S suit able for analytical and preparative purposes. Further, in the separati on of multiple forms of rabbit reticulocyte hexokinase, the HIC method was shown to be superior to RP-HPLC, making possible the efficient se paration of proteins with high molecular mass and their recovery in ac tive forms. The Toyopearl Phenyl 650 S column was also shown to be mor e efficient than the ion-exchange chromatographic media previously use d, allowing a quicker analysis of the multiple forms of rabbit reticul ocyte hexokinase under different biological conditions.