An accurate method to quantify ribulose bisphosphate carboxylase content in plant tissue

Abstract

ABSTRACTA method is described to accurately measure the content of ribulose bisphosphate carboxylase (RuBP carboxylase, EC 4·1.1·39) in plant tissues. This procedure, termed the internal standard method, involves extraction of the plant tissue (containing an unknown amount of 1H‐RuBP carboxylase) in a buffer containing a known amount of previously purified 3H‐RuBP carboxylase (internal standard). The rapid and efficient, single step copurification of 1H‐ and 3H‐RuBP carboxylases on the Mono Q column of the Fast Protein Liquid Chromatography System (FPLC), or by sucrose density gradient ultracentrifugation, allows the accurate estimation of the purification yield (3H in purified enzyme/3H in the extraction buffer). Knowing the amount of 1H‐RuBP carboxylase in the purified enzyme and the purification yield, one can calculate the concentration of 1H‐enzyme present in the plant tissue. This procedure overcomes some of the main constraints associated with the methods described in the literature: it takes into account the enzyme that is lost during the clarification of the protein extracts or during the isolation and purification processes; it is independent of the proteolysis that occurs in vitro by the action of cell proteases; it is not affected by the presence of RuBP carboxylase breakdown products; it is not influenced by any of the factors that control the catalytic activity or the activation state of the enzyme; and, it does not depend on the specificity of antigen‐antibody reactions.