A SIMPLE AND IMPROVED METHOD OF ISOLATION AND PURIFICATION FOR NATIVE OAT PHYTOCHROME

Abstract

A simple procedure for the isolation and purification of 124 kDa phytochrome (phyA) form etiolated Avena seedlings has been developed employing ammonium sulfate back-extraction. After solubilization of the ammonium sulfate precipitate (250 g/L) an additional ammonium sulfate fractionation with 17 g per 100 mL rather than column chromatography was performed. After several steps of the "washing-out" procedure with 100 mM phosphate buffer, phytochrome was solubilized in 10 mM phosphate buffer. The resulting phytochrome had a specific absorbance ratio (SAR = A666/ A280) ranging from 0.60 to 0.85. These values are equivalent to those of phytochrome preparations after hydroxylapatite chromatography-ammonium sulfate back-extraction. The total isolation-purification time was 8 h and yield of the chromoprotein was 50% higher than the yield using conventional techniques. The phytochrome preparation, after application to a Toyopearl HW-65S gel filtration column, produced very pure 124 kDa phyA with a specific absorbance ratio greater than 1.00. The spectral characteristics are identical to those described for the best of the highly purified native chromoprotein preparations.