A comparison between the coupled spectrophotometric and uncoupled radiometric assays for RuBP carboxylase

Abstract

The coupled spectrophotometric assay for RuBP carboxylase was compared with the conventional radiometric assay to assess the validity of its use in the measurement of initial and total activities in crude leaf extracts. At high magnesium concentrations both assays gave the same absolute values of initial and total activities, and resolved similarly the changes of total activity and activation state (ratio of initial to total activity) which occurred when the water status and light environment of leaves was altered prior to sampling. Although the magnesium concentration supporting the maximum rate of initial activity in soybean extracts was similar in the two assays, substantial differences of initial activity were observed at sub-optimal concentrations of magnesium. At low magnesium concentrations reaction rates in the spectrophotometric assay exhibited an initial phase of non-linearity which subsequently gave way to a linear rate. In contrast, reaction rates at low magnesium were linear from the time of initiation in the radiometric assay. Inclusion of EDTA in the reaction medium did, however, induce non-linear rates in the radiometric assay. The pre-addition of RUBP to extract immediately prior to dilution into the reaction medium did not eliminate the non-linearity in either assay system. The significance of these observations is discussed briefly in relation to the use of the spectrophotometric assay.