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 mumol per 100 g of wet tissue and used for arylsulfatase B (ASB) activity determination. Two HPLC methods of separation and quantitation of the reaction product were described: (1) an original gradient elution method which makes it possible to determine the reaction product when only partially purified ASB was used and additional uridine derivatives were formed during incubation; (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 < or = 3%. Using the gradient elution method we have found that UDP-GalNAc-4-S was hydrolysed by bovine arylsulfatase B1 most efficiently at pH 5.0 and concentration 0.5 mM with K(m) = 85 microM.
Leznicki
A J
AJ
Department of Biochemistry, Nicholas Copernicus University, Torun, Poland.
Bialkowski
K
K
D000818:Animals; D002417:Cattle; D002645:Chickens; D002803:Chondro-4-Sulfatase / Q000032:analysis* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006868:Hydrolysis; D007700:Kinetics; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000031:analogs & derivatives* / Q000737:chemistry
C034984:uridine diphosphate-N-acetylgalactosamine 4-sulfate; D014536:Uridine Diphosphate N-Acetylgalactosamine; D002803:Chondro-4-Sulfatase