The physicochemical properties of peroxidase isolated from cotton leaves were investigated. The optimal pH value for exhibiting activity was 4.7; temperature, 30°C. The Michaelis constant was 2.3 mM. Cottonleaf peroxidase has a very high affinity for benzidine.Peroxidase is one of the most widely distributed plant enzymes and has been studied in horseradish, wheat, alfalfa, peas, barley, potatoes, etc.[1].All plant organs contain peroxidase. This is indicative of its universal functions. Therefore, we isolated peroxidase from various cotton organs: stem, root, pods, integument, fiber, and aging leaves in various vegetative stages. Peroxidase isolated from cotton leaves during extensive opening of the pods was most active.The specific activity of peroxidase (U/mg) isolated from various cotton organs was 9.1 from stem; 8.9, root; 7.8, pods; 3.1, integument; 2.05, fiber; and 120, leaves.Gel filtration over TSK-HW-65 gel produced three fractions, of which the second fraction had peroxidase activity. Further purification by ion-exchange chromatography over DEAE-cellulose gave a purified peroxidase activity of 840 U/mg (Fig. 1). This fraction was used in the investigations.It is important in the study of enzymes to consider the optimal conditions needed for reactions to occur. These include thermal stability, substrate specificity, isoelectric points, and optimum pH.Proteins with peroxidase activity at pH 3.5-4.7 were determined by isoelectric focusing on PAAG-plates containing ampholines in the pH range 3.5-9.5 (Fig. 2).Plant isoperoxidases consist of acidic and basic enzyme forms. The cotton-leaf peroxidase that we obtained was mainly the acid forms. By reviewing the literature data and ours [2, 3], it can be seen that acidic isoperoxidases have a higher specific activity for benzidine, o-phenylendiamine, luminol, ammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and diaminobenzidine whereas basic ones are more specific for guaiacol, phenyl-antipyrine, and pyrogallol. Cotton-leaf peroxidase has the highest affinity for benzidine, o-dianisidine (ODN), and ABTS (Table 1).The enzyme activity as a function of pH is a superposition of two pH dependencies. These are intrinsic to the substrate conversion and conformational changes of the enzyme globule. Standard conditions for measuring the peroxidase activity are known. A more appropriate characteristic of isoperoxidase is the pH of optimum stability, which is independent of the substrate, is determined by the nature of the enzyme, and, as a rule, is close to the p i of the protein.Data have been published [2] indicating that the enzyme has different optimal pH values depending on the source and is active at pH values from 4 to 8. It is also known that peroxidase isoforms from a single plant are active at different pH values [1]. Local pH changes in the cell probably regulate the inclusion of some isoenzymes or others in the oxidation of phenolic compounds. This justifies the existence of multiple peroxidase forms.Various buffers at pH 3.6-9 were used to ...
