Some observations on the role of copper ions in the reduction of phosphomolybdate by ascorbic acid and their application in the determination of inorganic orthophosphate

Abstract

The reduction of phosphomolybdate to a blue compound by ascorbic acid under the conditions of Lowry and Lopez (1946) is inhibited by extracts of a rumen microorganism and by liver and brain extracts. Strong inhibitions are observed in the presence of micromolar concentrations of sodium K, diethyldithiocarbamate and ethylenedlaminetetra-acetic acid and in the presence of millimolar concentrations of cysteine. At low concentrations (e.g. 2 x 10-6[image]), cupric ions cause a marked acceleration of the rate of color development. At higher concentrations (1.7 x 10-4 [image]) the final color attained is also increased, and above this concentration a marked color appears in the absence of phosphate. No comparable effects are observed with Mg++, CO++, Ni++, Mn++, Fe++, Fe+++ or Sn++ ions. Cuprous ions reduce phosphomolybdate to the blue complex under the conditions for phosphate assay. It is suggested that copper ions act as electron carriers in the reduction of phosphomolybdate by ascorbic acid and that normally sufficient copper is present as impurity in the reagents. In the presence of metal-binding agents, including those occurring in tissues, phosphomolybdate reduction is inhibited as a result of metal-binding action. The addition of 4.2 x 10-6[image] copper ions to the system overcomes the effect of such concentrations of metal-binding agents as are likely to be met with in normal practice. A suitable modification to the procedure of Lowry and Lopez is described. By using a somewhat higher concentration (1.7 x 10-4 [image]) of copper ions, the sensitivity of the method can be increased by 50% though the timing of operations becomes more critical under these conditions. Concentrations above 3.3 x 10-4 [image] interfere with the method.