Purification of yeast hexokinase isoenzymes using affinity chromatography and chromatofocusing

A new procedure has been devised for the rapid isolation of yeast hexokinase isoenzymes PI and PII, giving specific activities comparable to those obtained after conventional purification. Hexokinases were bound to d-glucosamine, which had been coupled to CH Sepharose 4B using 6-aminohexanoic acid as a spacer. An ATP/ d-glucose/MgCl 2 solution was used for elution. After concentration with DEAE-Sephacel, isoenzymes were separated by chromatofocusing. Hexokinase PI gave a single band on polyacrylamide gel electrophoresis, whereas one minor foreign band was seen for hexokinase PII.