. Author manuscript; available in PMC: 2010 Jul 26.

Published in final edited form as: Biochemistry. 2000 May 23;39(20):6219–6227. doi: 10.1021/bi992464j

Table 2.

Analyses of Various Forms of Hex B: Heat Stability, Kinetic parameters (MUG), and Ability to Specifically Bind a CNAG Hex Affinity Column

isozyme	t_1/2 (min) 60 °C	SA^a	K_m^b (mM)	Hex B (ng/assay)	V_max^b (nmol/h)	k_cat × 10⁻³^c (mol/mol h)	k_cat × 10⁻³/K_m	% binding to CNAG^d
Hex B (mature)	13 ± 1^e	10.1 ± 0.5	0.69 ± 0.09	0.78	11.3 ± 0.5	1900 ± 100	2700 ± 600	75 ± 5
pro-Hex B	14 ± 1	10.5 ± 0.6	0.71 ± 0.07	0.70	10.6 ± 0.3	2000 ± 100	2700 ± 500	72 ± 6
pro-Hex B-His₆	17 ± 1	13.8 ± 0.8	0.66 ± 0.07	0.51	10.0 ± 0.3	2500 ± 100	3800 ± 600	71 ± 4
pro-Hex B- (Arg²¹¹Lys)-His₆	ND^h	0.007 ± 0.0004	~8^f	1600	~64^f	~5^f	0.62 ± 0.02^g	7 ± 1

Specific activity, nmoles (MU) h⁻¹ ng⁻¹ (Hex B), at 1.6 mM MUG.

K_m values are given in mM (MUG), and V_max values are given as nmol of MU h⁻¹, with the standard error reported as “±”. Note that V_max values are those derived from Figure 4 and have not been normalized for the amount of Hex B protein used in the assay (given in column 5), i.e., they are not “specific activities” at V_max.

k_cat values are the maximum moles of MU released h⁻¹ (mol of various forms of purified Hex B protein)⁻¹, which are proportional to the specific activities at V_max. They all assume an M_r for Hex B of 130 000.

Percent of Hex protein binding from three independent experiments: 100 × μg of protein-eluted (with δ-lactone) × (10 μg of protein loaded)⁻¹ (on a CNAG minicolumn).

Standard error of at least three determinations.

Accuracy of the V_max, K_m, and k_cat values could not be ensured due to the limited substrate solubility.

The k_cat/K_m ratio was accurately calculated from 130 000× the slope of the best-fit straight line of [MUG] (0–1.25 mM) versus (nmol of MU) h⁻¹ [ng of pro-Hex B-(Agr²¹¹Lys)-His₆ used in each assay]⁻¹.

ND, not determined.