Influence of pigments and pH of urine on the determination of N-acetyl-beta-D-glucosaminidase activity with 2-methoxy-4-(2'-nitrovinyl)-phenyl-N-acetyl-beta-D-glucosaminide.

Abstract

The influence of urinary pigments and urine pH on the spectrophotometric determination of N-acetyl-beta-D-glucosaminidase (NAG; EC 3.2.1.30) activity with 2-methoxy-4-(2'-nitrovinyl)-phenyl-N-acetyl-beta-D-glucosaminide as a substrate was studied. The investigation was performed with human and rabbit urine samples. It was found that alkaline urine pH values influenced NAG activity in two ways: 1) NAG activity decreased due to enzyme instability with pH increase, and 2) NAG activity increased because of the contribution of urinary pigments to absorbance of 2-methoxy-4-(2'-nitrovinyl)-phenol (MNP) at 505 nm. It was shown that besides the maximum (I) in the range of 350-360 nm of the absorption spectra of alkaline urine, there was a maximum (II) in the range of 380-460 nm. With the increase of pH, maximum II was shifted toward higher wavelengths and contributed to MNP absorption (5-90%). On the other hand, the maximum of MNP absorption was shifted toward lower wavelengths (495-400 nm) with increasing pH. Two procedures to eliminate the influence of urinary pigments are presented. The justification of applying a correction to the values of NAG activity in human and rabbit urine (a model system for studying the toxic effects of cadmium) was discussed.

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