Standardization of α-L-iduronidase enzyme assay with Michaelis-Menten kinetics

Abstract

The lack of methodological uniformity in enzyme assays has been a long-standing difficulty, a problem for bench researchers, for the interpretation of clinical diagnostic tests, and an issue for investigational drug review. Illustrative of the problem, α-L-iduronidase enzyme catalytic activity is frequently measured with the substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide); however, final substrate concentrations used in different assays vary greatly, ranging from 25 μM to 1425 μM (Km ≈ 180 μM) making it difficult to compare results between laboratories. In this study, α-L-iduronidase was assayed with 15 different substrate concentrations. The resulting activity levels from the same specimens varied greatly with different substrate concentrations but, as a group, obeyed the expectations of Michaelis-Menten kinetics. Therefore, for the sake of improved comparability, it is proposed that α-L-iduronidase enzyme assays should be conducted either (1) under substrate saturating conditions; or (2) when concentrations are significantly below substrate saturation, with results standardized by arithmetic adjustment that considers Michaelis-Menten kinetics. The approach can be generalized to many other enzyme assays.

Keywords: 4-methylumbelliferyl; Hurler syndrome; IDUA; Lineweaver–Burk plot; MPS; MU; Michaelis–Menten kinetics; mucopolysaccharidoses; mucopolysaccharidosis; α-L-iduronidase; α-L-iduronidase enzyme assay.

Copyright © 2013 Elsevier Inc. All rights reserved.

Figures

Figure 1. Michaelis-Menten analysis of α-L-iduronidase enzyme activity in liver from normal heterozygous mice (idua−/+)

Each point is the mean of 3 mice. The line indicates the approximate plateau that defines Vmax.

Figure 2. Lineweaver-Burk analysis of α-L-iduronidase enzyme activity in liver from normal heterozygous mice (idua−/+)

Each point is the mean value from 7 mice.

Figure 3. α-L-iduronidase enzyme assay with a final substrate concentration of 180 μM is linear through 50 min

Five groups of reactions were quenched at different time points (10, 20, 30, 40, and 50 min), and the fluorescence was measured.