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[4] Enzymic microdetermination of d-glucose, d-fructose, d-gluconate, 2-keto-d-gluconate, aldehyde, and alcohol with membrane-bound dehydrogenases

1982; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(82)89006-x

1557-7988

Minoru Ameyama,

Pancreatic function and diabetes

Enzymic microdetermination of sugars is based on measuring reduced pyridine nucleotides, such as alcohol, with nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase and D-glucose with a coupling reaction composed of hexokinase, hexosephosphate isomerase, and D-glucose- 6-phosphate dehydrogenase. Sometimes the reduced pyridine nucleotide is amplified by dyes, such as nitro blue tetrazolium, and also allows activity measurement in the visible region. For enzymic microdetermination of various substrates, the use of individual membrane-bound microbial dehydrogenases is recommended. The acetic acid bacteria have many dehydrogenases on the outer surface of cytoplasmic membrane, and these catalyze the oxidation of various carbohydrates and accumulate a large amount of oxidation product in the culture medium, for instance, D-gluconate, 2-keto-D-gluconate, 5- keto-D-fructose, 2,5-diketo-D-gluconate, acetate, dihydroxyacetone, 5-keto-D-gluconate, and L-sorbose. Enzymic microdetermination of substrate utilizes the method of Wood using ferricyanide as an electron acceptor with some modifications. The method is based on the initial reduction rate. This chapter focuses on the ferricyanide method because to its simplicity for routine use. With 2,6-dichlorophenolindophenol, the molecular extinction coefficient is variable with pH and makes the assay less reliable for quantitative determination.