IMPROVED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR N-ACETYLGALACTOSAMINE-4-SULFATE SULFATASE (ARYLSULFATASE-B) ACTIVITY DETERMINATION USING URIDINE DIPHOSPHO-N-ACETYLGALACTOSAMINE-4-SULFATE

UDP-N-acetylgalactosamine-4-sulfate (UDP-GalNAc-4-S) was isolated from hen oviduct (isthmus) with a yield of 31 mu mol per 100 g of wet tiss ue and used for arylsulfatase B (ASB) activity determination. Two HPLC methods of separation and quantitation of the reaction product were d escribed: (1) an original gradient elution method which makes it possi ble to determine the reaction product when only partially purified ASB was used and additional uridine derivatives were formed during incuba tion; (2) an improved, fast isocratic elution method which may be used in the case of purified ASB preparations, devoid of other nucleotide hydrolysing enzymes. For both methods the detection limit was 0.1 nmol of product with standard error of determination less than or equal to 3%. Using the gradient elution method we have found that UDP-GalNAc-4 -S was hydrolysed by bovine arylsulfatase Fl most efficiently at pH 5. 0 and concentration 0.5 mM with K-m=85 mu M. (C) 1997 Elsevier Science B.V.