Simple, rapid spectrophotometry of urinary N-acetyl-beta-D-glucosaminidase, with use of a new chromogenic substrate.

Abstract

We have developed a new spectrophotometric assay for urinary N-acetyl-beta-D-glucosaminidase (NAGase) with use of sodio m-cresolsulfonphthaleinyl N-acetyl-beta-D-glucosaminide (MCP-NAG). MCP-NAG was synthesized from acetochloro-glucosamine and m-cresolsulfonphthalein (MCP) in four steps. MCP-NAG reacts well with NAGase (Km = 0.41 mmol/L) and is highly water soluble. The absorption maximum and molar absorptivity of the aglycone MCP are 580 nm and 40 670, respectively. Spectral overlap of interfering substances at 580 nm is almost negligible, so that the urine blank can be omitted from the assay procedure. The high molar absorptivity of MCP gives sufficient analytical sensitivity at a reaction time of 15 min. The correlation between the MCP-NAG method (y) and the fluorimetric method (x) involving 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide is represented by the equation y = 0.995x - 0.669 (r = 0.991). Thus, the present method provides practical advantages over conventional methods, for use in the routine laboratory.