Comparative study of the efficacies of nine assay methods for the dextransucrase synthesis of dextran

摘要

A comparative study of nine assay methods for dextransucrase and related enzymes has been made. A relatively widespread method for the reaction of dextransucrase with sucrose is the measurement of the reducing value of D-fructose by alkaline 3,5-dinitrosalicylate (DNS) and thereby the amount of D-glucose incorporated into dextran. Another method is the reaction with ~(14)C-sucrose with the addition of an aliquot to Whatman 3MM paper squares that are washed three times with methanol to remove ~(14)C-D- fructose and unreacted ~(14)C-sucrose, followed by counting of ~(14)C-dextran on the paper by liquid scintillation counting (LSC). It is shown that both methods give erroneous results. The DNS reducing value method gives extremely high values due to over-oxidation of both D-fructose and dextran, and the ~(14)C-paper square method gives significantly low values due to the removal of some of the ~(14)C-dextran from the paper by methanol washes. In the present study, we have examined nine methods and find two that give values that are identical and are an accurate measurement of the dextransucrase reaction. They are (1) a ~(14)C-sucrose/dextransucrase digest in which dextran is precipitated three times with three volumes of ethanol, dissolved in water, and added to paper and counted in a toluene cocktail by LSC; and (2) precipitation of dextran three times with three volumes of ethanol from a sucrose/dextransucrase digest, dried, and weighed. Four reducing value methods were examined to measure the amount of D-fructose. Three of the four (two DNS methods, one with both dextran and D-fructose and the other with only D-fructose, and the ferricyanide/arsenomolybdate method with D-fructose) gave extremely high values due to over-oxidation of D-fructose, D-glucose, leucrose, and dextran.