[94] Triosephosphate isomerase from human erythrocytes
1975; Academic Press; Linguagem: Inglês
10.1016/s0076-6879(75)41096-5
ISSN1557-7988
Autores Tópico(s)Hemoglobin structure and function
ResumoThis chapter describes the assay method, purification procedure, and properties of triosephosphate isomerase from human erythrocytes. Triosephosphate isomerase activity can be measured in either direction by coupling to the appropriate dehydrogenase and following the rate of oxidation or reduction of NADH or NAD at 340 nm. Arsenate, a necessary component of the assay, is a competitive inhibitor of triosephosphate isomerase, and corrections for this inhibition must be taken into consideration. The two coupling enzymes are both commercially available as crystalline suspensions essentially free of triosephosphate isomerase activity. However, since triosephosphate isomerase is inhibited by ammonium sulfate, the coupling enzymes are dialyzed prior to use. The isolation of triosephosphate isomerase from the human erythrocytes is described. Crystalline human triosephosphate isomerase can be resolved into three forms by electrophoresis or isoelectric focusing. For preparative purposes the enzyme is electrofocused in 1% narrow range (pH 5–7) Ampholines for 92 hr at 600 V. The enzyme is not homogeneous after the second chromatographic step on DEAE-cellulose and is contaminated with a protein of large molecular weight. Human triosephosphate isomerase is stable when stored in 0.80 saturated ammonium sulfate at 0–4°. Human triosephosphate isomerase is rapidly inactivated by the substrate analog, chloroacetol phosphate, by the selective esterification of a single essential glutamyl γ-carboxylate per catalytic subunit.
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