ABSTRACT
Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.
Subject(s)
Cholestanols/chemistry , Plant Growth Regulators/chemistry , Steroids, Heterocyclic/chemistry , Animals , Antigens, Plant/analysis , Antigens, Plant/immunology , Brassinosteroids , Cholestanols/analysis , Cholestanols/immunology , Cross Reactions , Immune Sera/isolation & purification , Immunoenzyme Techniques , Plant Growth Regulators/immunology , Rabbits , Steroids, Heterocyclic/analysis , Steroids, Heterocyclic/immunologyABSTRACT
The horseradish peroxidase (HRP) conjugates with the sheep antirabbit antibodies and cortisol (COR) or progesterone (PROG) containing 9 to 40 steroid molecules per HRP molecule were synthesized. In aqueous media all the conjugates have lower catalytic activity in the o-phenylenediamine oxidation than the native enzyme. In reversed Aerosol OT micelles in heptane the HRP-COR and HRP-PROG conjugates containing 12 and 9 steroid molecules, respectively, have catalytic constants 2.6 and 2.7 times higher than the unmodified enzyme. The influence of the HRP hydrophobisation and its inactivation on the course of modification on catalytic properties of the enzyme is discussed.
Subject(s)
Horseradish Peroxidase/chemistry , Hydrocortisone/chemistry , Progesterone/chemistry , Catalysis , Horseradish Peroxidase/immunology , Hydrocortisone/immunology , Kinetics , Micelles , Oxidation-Reduction , Progesterone/immunology , WaterABSTRACT
Catalase conjugates with 3, 7, 9 and 42 progesterone molecules were obtained by the reaction between the enzyme and N-oxy-succinimide ether of 3-0-carboxymethyloxime of progesterone. The enzyme modified by 42 progesterone molecules is effective in o-dianisidine oxidation by hydrogen peroxide and has a kcat/KM value of 512 M-1 s-1. The catalase conjugates with 3, 7 and 9 progesterone molecules exhibit a high activity during o-dianisidine oxidation by cumene hydroperoxide. The activity of conjugates is higher than that of the native non-modified enzyme in the same reaction. The maximum effectiveness was observed for catalase modified by 7 progesterone molecules. This conjugate is characterized by kcat/KM of 99,000 M-1 s-1 at 30 degrees C. The effect of the degree of enzyme modification on the kinetic parameters of o-dianisidine oxidation by H2O2 and cumene hydroperoxide is discussed.
