Mini-chromatofocusing of plant and fungal polyphenoloxidases

Mini-chromatofocusing was used to estimate the isoelectric points of mung bean polyphenoloxidase (5.4), broad bean polyphenoloxidase (5.4-5.7), and mushroom tyrosinase (4.5) in crude and purified samples. Using 1 to 2 ml of Polybuffer exchanger and 20 to 25 ml of Polybuffer 74, isoelectric points for the above enzymes were obtained in less than 2 h. Using this technique, isoelectric points for beta-lactoglobulin (4.5), carbonic anhydrase (6.4), soybean trypsin inhibitor (4.1), myoglobin (6.9), and bovine serum albumin (4.8) were obtained and compared to existing literature values. Mini-chromatofocusing provides a rapid estimation for isoelectric points of proteins and enzymes and may be a useful alternative to conventional methods for determination of isoelectric points.