Studies on the use of Sepharose-N-Aminohexanoyl-Glucosamine for purification of rabbit red blood cell hexokinase

N-acetyl glucosamine is a competitive inhibitor (K _t= 0.7 mM) of red blood cell hexokinase with respect to glucose. This property has been utilized for the purification of hexokinase by means of Sepharose-TV-aminohexanoyl-glucosamine. Studies with this matrix have proved that ionic strength and pH play a very important role in the binding of hexokinase to the affinity column. Therefore their control is essential in order to minimize nonspecific binding and to maximize the purification. Methods for rejuvenation of columns, the effect of protein concentration, and the nature of the binding are also discussed in this paper.