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

A method is described to accurately measure the content of ribulose bisphos phate carboxylase (RuBP carboxylase, EC 4.1.1.39) in plant tissues. This pr ocedure, termed the internal standard method, involves extraction of the pl ant tissue (containing an unknown amount of H-1-RuBP carboxylase) in a buff er containing a known amount of previously purified H-3-RuBP carboxylase (i nternal standard). The rapid and efficient, single step copurification of H -1- and H-3-RuBP carboxylases on the Mono Q column of the Fast Protein Liqu id Chromatography System (FPLC), or by sucrose density gradient ultracentri fugation, allows the accurate estimation of the purification yield (H-3 in purified enzyme/H-3 in the extraction buffer). Knowing the amount of H-1-Ru BP carboxylase in the purified enzyme and the purification yield, one can c alculate the concentration of H-1-enzyme present in the plant tissue. This procedure overcomes some of the main constraints associated with the method s described in the literature: it takes into account the enzyme that is los t during the clarification of the protein extracts or during the isolation and purification processes; it is independent of the proteolysis that occur s in vitro by the action of cell proteases; it is not affected by the prese nce 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.