Pócsi I, Taylor S A, Richardson A C, Aamlid K H, Smith B V, Price R G
Biochemistry Department, King's College London, London, U.K.
Clin Chem. 1990 Nov;36(11):1884-8.
We describe a new chromogenic substrate and assay for determining N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30; NAG) activity in normal and pathological human urine. The novel substrate, ammonium 5-[4-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-3- methoxyphenylmethylene]-2-thioxothiazolidin-4-one-3-ethan oate (VRA-GlcNAc), is prepared by condensation of vanillyl N-acetyl-beta-D-glucosaminide and rhodanine-3-ethanoic acid. The phenol released by enzyme action has an intense absorption peak at 492 nm (epsilon = 37,000 L.mol-1.cm-1). The Km for NAG in urine was 0.5 mmol/L at pH 4.5. The substrate solution may be stored at 4-7 degrees C for one week without any increase in the substrate blank. For optimal assay conditions, we used a substrate concentration of 3 mmol/L and a 15-fold sample dilution at pH 4.5-5.0, with measurement at 505 nm, not significantly different from that at 492 nm. The sensitivity of the assay with VRA-GlcNAc as substrate is twice that of the MNP-GlcNAc [2-methoxy-4-(2'-nitrovinyl)-phenyl GlcNAc] procedure (Clin Chim Acta 1982;124:195-204) and can detect low amounts of minor NAG isoenzymes. This assay is an improvement on previously available colorimetric methods and could be easily incorporated into kits based on either an enzyme calibrant or the molar absorptivity of the phenol.
我们描述了一种用于测定正常和病理性人尿液中N-乙酰-β-D-氨基葡萄糖苷酶（EC 3.2.1.30；NAG）活性的新型显色底物及检测方法。新型底物5-[4-(2-乙酰氨基-2-脱氧-β-D-吡喃葡萄糖氧基)-3-甲氧基苯亚甲基]-2-硫代噻唑烷-4-酮-3-乙酸铵（VRA-GlcNAc）是通过香草基N-乙酰-β-D-氨基葡萄糖苷与罗丹宁-3-乙酸缩合制备而成。酶作用释放出的苯酚在492 nm处有一个强吸收峰（ε = 37,000 L·mol⁻¹·cm⁻¹）。尿液中NAG在pH 4.5时的Km为0.5 mmol/L。底物溶液可在4 - 7℃下储存一周，底物空白值无任何增加。为获得最佳检测条件，我们使用3 mmol/L的底物浓度，在pH 4.5 - 5.0下将样品稀释15倍，并在505 nm处进行测量，该波长下的测量结果与492 nm处的结果无显著差异。以VRA-GlcNAc为底物的检测方法的灵敏度是MNP-GlcNAc [2-甲氧基-4-(2'-硝基乙烯基)-苯基GlcNAc]方法（《临床化学学报》1982年；124:195 - 204）的两倍，并且能够检测到少量的次要NAG同工酶。该检测方法是对先前可用的比色法的改进，并且可以很容易地整合到基于酶校准物或苯酚摩尔吸光系数的试剂盒中。
