An ultraspecific micromethod for the determination of d-glucose

✍ Scribed by M.Y. Kamel; R.R. Hart; R.L. Anderson

• Publisher: Elsevier Science
• Year: 1967
• Tongue: English
• Weight: 199 KB
• Volume: 18
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(67)90010-3

✦ Synopsis

Of the plethora of published methods for the determination of n-glucose, those which have the advantage of being both highly specific and sensitive have employed glucose oxidase (1) or hexokinase (2) as assay reagents.2 Although these methods have proved highly useful in many applications, they are of limited value when n-glucose must be determined in the presence of relatively high concentrations of certain other sugars such as n-mannose, as was encountered in a recent investigation of n-mannose metabolism (3). One of the disadvantages of glucose oxidase arises from the fact that it has a significant activity on n-mannose (4). Consequently, when the ratio of n-mannose to n-glucose in a mixture is about 106 to 1 or greater, the oxidation of n-glucose becomes masked and cannot be accurately measured. Another disadvantage of glusose oxidase arises from the use of peroxidase as a coupling enzyme to measure the H202 produced in the reaction. Thus, the measurements are subject to interference from competing electron donors such as glutathione or ascorbic acid, which are frequently either present in biological materials or are necessary additions to systems on which the assays for n-glucose must be made. The main disadvantage of the hexokinase/glucose-6-phosphate dehydrogenase assay for n-glucose is the lack of specificity of hexokinase, which phosphorylates n-mannose and n-fructose readily; thus, at a high n-mannose to n-glucose ratio, the values for n-glucose are generally low due to the competition for ATP by n-mannose. Also, n-mannose 6-phosphate inhibits hexokinase (5).

The difficulties encountered by the use of glucose oxidase or hexokinase as assay reagents for n-glucose can be circumvented by the use of a highly stereospecific D-ghrcokinase recently purified from Aerobacter