Table 3. Rates and Extents of NO Generation on Hydrolysis of 9a and 9b under Catalysis by N-Acetylglucosaminidases from Both Jack Bean (JB) and Human Placenta (HP)^a.

		moles produced per mole of glycoside
compd	enzyme	[NO]	[NO2−]	[NO + NO2−]	k (s−1)
9a	JB	1.26	0.05	1.31	6.2 × 10−4
9a	HP	1.73	0.24	1.97	4.1 × 10−4
9b	JB	1.72	0.12	1.84	3.1 × 10−4
9b	HP	1.69	0.36	2.05	3.6 × 10−4

^aAll reactions were run at 37 °C in 0.1 M phosphate, pH 7.4, containing 0.05 mM diethylenetriaminepentaacetic acid. Jack bean (JB) enzyme was present at 0.11 units/mL, while its human placenta (HP) counterpart was added at 0.044 units/mL. Substrates were added at the following initial concentrations: 9a, 0.10 mM; 9b, 0.16 mM; 5a, 0.09 mM. NO production was followed by chemiluminescence detection until no more was evolved. Nitrite formed during hydrolysis was measured in the remaining solution by the colorimetric Griess assay. Rate constants were cleanly first order, as determined by ultraviolet spectrophotometry; wavelengths followed were 227 nm for 9a and 5a but 256 nm for 9b. Absorbance changes in the 5a incubations were negligible under the conditions employed. The enzymes used were confirmed to be fully active by means of control incubations with the manufacturer’s recommended substrate, 1-(p-nitrophenyl)-2-deoxy-2-(N-acetyl)-β-d-glucosamine.