APPLICATION OF HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY TO DETERMINATION OF MODIFIER ACTIVITY IN ALPHA-LACTALBUMIN AND OTHER PROTEINS

High-performance liquid chromatography has been applied to determinati on of modifier activity in alpha-lactalbumin (alpha-LA). An amino-bond ed column separates uridine diphosphate (UDP) (product), UDPgalactose (substrate), and uridine monophosphate (UMP). From an aliquot of the s ame sample, a column for carbohydrate analysis separates lactose (the other product) and galactose-1,2 cyclic phosphate (Gal-c-P). Nucleotid e peaks are detected by measurement of A(262) and those of carbohydrat e by H-3 counting, the isotope originating from UDP-galactose-H-3. A p H of 6.3 was taken as optimal for production of UDP since, at this lev el, the unwanted side reaction is minimized, by which UMP and Gal-c-P are formed. Thus, the conservation of substrate so effected may have c ontributed to an enhanced production of UDP. The reaction by which UDP and lactose are produced was linear for 120 min, as followed by UDP f ormation, but it continued to at least 300 min. Production of lactose was equivalent to that of UDP, when alpha-LA was the modifying protein . From a survey of seven other proteins, only lysozyme and ovalbumin s howed ability to produce UDP. However, failure of the last two protein s to produce lactose indicates absence of modifier activity and demons trates the need for monitoring both products. (C) 1995 Academic Press, Inc.