[47] Glucose-6-phosphate Dehydrogenase from Human Erythrocytes

Philip Cohen, Michael A. Rosemeyer

    Research output: Contribution to journalArticle

    34 Citations (Scopus)

    Abstract

    This chapter describes the assay method, purification procedure, and properties of glucose-6-phosphate dehydrogenase from human erythrocytes. The activity of glucose-6-phosphate dehydrogenase is measured in the presence of excess glucose 6-phosphate and NADP by the increase in absorbance at 340 nm, which corresponds to the formation of NADPH. The reaction goes to completion since the product 6-phosphogluconolactone is rapidly hydrolyzed to 6-phosphogluconate. This hydrolysis is not a rate-limiting step in the standard assay. The maximum reaction velocity and Km are dependent on the ionic strength of the buffer, and are influenced by the particular ions present. For the purification procedure, blood is stored at 0–4° in acid-citrate-dextrose to prevent clotting, and the cells are centrifuged after each wash at 3000 g for 15 rain, and the supernatant is sucked off. A summary of the purification is also tabulated. On correction, the freshly prepared enzyme has a specific activity of 220 units/mg. This value declines to 150 nnits/mg over a period of 2 weeks. The preparation is homogeneous by the criteria of electrophoresis on starch gel at pH 8.6, 5 and on cellulose acetate at pH 7.5, and on polyacrylamide gels in the presence of sodium dodecyl sulfate. Some general properties of the enzyme and of the amino acid composition are summarized.

    Original languageEnglish
    Pages (from-to)208-214
    Number of pages7
    JournalMethods in Enzymology
    Volume41
    Issue numberC
    DOIs
    Publication statusPublished - 1975

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    Glucosephosphate Dehydrogenase
    Purification
    Erythrocytes
    NADP
    Assays
    Starch Gel Electrophoresis
    Glucose-6-Phosphate
    Rain
    Enzymes
    Ionic strength
    Electrophoresis
    Sodium Dodecyl Sulfate
    Starch
    Osmolar Concentration
    Hydrolysis
    Buffers
    Blood
    Gels
    Ions
    Amino Acids

    Cite this

    Cohen, Philip ; Rosemeyer, Michael A. / [47] Glucose-6-phosphate Dehydrogenase from Human Erythrocytes. In: Methods in Enzymology. 1975 ; Vol. 41, No. C. pp. 208-214.
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    [47] Glucose-6-phosphate Dehydrogenase from Human Erythrocytes. / Cohen, Philip; Rosemeyer, Michael A.

    In: Methods in Enzymology, Vol. 41, No. C, 1975, p. 208-214.

    Research output: Contribution to journalArticle

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