Assay of beta-N-acetylhexosaminidase isoenzymes in urine by means of determination of their activation energy without removing endogenous low-molecular-mass components.
The determination of the activation energy of beta-N-acetylhexosaminidase (Hex, EC 3.2.1.52), using 3,3'-dichlorophenylsulfonphthaleinyl-N-acetyl-beta-D-glucosaminide as substrate, allows its isoenzyme composition to be evaluated in different biological specimens. However, in the analysis of urine samples, it is necessary first to remove the endogenous low-molecular-mass components, as these provoke an over-estimation of the activation energy of the Hex and, consequently, of the relative proportion of Hex B isoenzyme. The study of this interference has allowed urea to be characterised as the only urinary metabolite that is responsible, and to establish a mathematical expression for the correction, in relation to the endogenous urea concentration, of the activation energy of the Hex obtained experimentally in samples of native urine. The results thus obtained for the isoenzyme composition of urinary Hex are similar to those found using an electrophoretic separation procedure.
Laboratorio Central, Hospital General de Galicia-Clínico Universitario, Santiago de Compostela, Spain.
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LF
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Tutor
D004789:Enzyme Activation; D054819:Hexosaminidase B; D006801:Humans; D007527:Isoenzymes / Q000652:urine*; D016014:Linear Models; D008433:Mathematics; D008970:Molecular Weight; D013816:Thermodynamics; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry* / Q000652:urine*
D007527:Isoenzymes; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases