Purification and Characterization of Isoperoxidases Elicited by Aspergillus flavus in Cotton Ovule Cultures.

Two anionic isoperoxidases were isolated from media of Aspergillus flavus-inoculated cotton (Gossypium hirsutum L.) ovule cultures and purified about 150-fold to apparent homogeneity by treatment with Cell Debris Remover and ion exchange chromatography on Accell QMA medium. These isoperoxidases were present in noninoculated cotton ovule cultures at low levels. The major activity peak (B) represented 90% of the recovered peroxidase activity and was electrophoretically homogeneous. The minor activity peak (A) was about 95% pure. Isoelectric focusing analysis showed that B was greater than 95% pure with respect to other peroxidase isozymes, while the enzyme in A was about 90% isozymically pure. Each isoperoxidase displayed a molecular mass of 56 kilodaltons by interpolation from denaturing gel electrophoresis. The B isozyme displayed a molecular mass of 55 kilodaltons by gel filtration chromatography. The pH optima for the cotton ovule isoperoxidases were similar, 5.0 for isozyme A and 6.0 for isozyme B. The isoelectric points for isozymes A and B were 4.2 and 4.4, respectively. Eugenol, guaiacol, and 3,3',5,5'-tetramethylbenzidine were good electron donor substrates, whereas 4-aminoantipyrine was a poor substrate. The absorption spectrum of the material in B revealed a major peak at 400 nanometers and a minor peak at 280 nanometers. The molar extinction coefficient at 400 nanometers (pH 7.0) was calculated to be 1.07 x 10(5) per square centimeter per mole. Amino acid analysis of isozyme B confirmed the acidic nature of this protein and identified a number of similarities to the anionic peroxidases from tobacco and potato. This glycoprotein was found to contain 12 to 14% sugar (by weight), mainly in the form of galactose and mannose.