Partial Purification of a Cell-Division Factor from Peas

Abstract

A highly active kinetin-like preparation was obtained from peas. About 7000 liters of blanching water used to process 25 tons of fresh peas were concentrated at low temperature and spray-dried to yield 99 kg of solids with biological activity corresponding to 3-6 [mu]g equivalents of kinetin per g in bloassays with tobacco tissue cultures. A total 14 kg of solids purified by repeated ion exchange resin chromatography, solvent extraction and Ag+ precipitation yielded 7.2 mg of product which when bioassayed in the range from 1-25 [mu]g/liter was at least as active as kinetin. The active material is organic. It is soluble in dilute acid or base, methanol, ethanol, butanol and partially in CHCl3. It is amphoteric and is precipitated by Ag+ even in strong acid. Its biological activity is destroyed by refluxlng 24 hours with 6 N HC1 at about 100[degree], but only partially lost by heating in 6 NNaOH. These properties together with UV absorption typical of purines and high biological activity point to an active purine derivative. The active material, unlike kinetin, is insoluble in ether, and the 2 are separable by chromatography. The presence of more than 1 active substance was indicated by biological activity in both CHCl3 soluble and insoluble fractions. When the 7.2 mg final product was refractionated on Dowex 1 Cl-, 90% of the total UV absorption was in a single peak which was biologically active, and the remaining 10% was distributed in 4 peaks which were inactive. A biologically inactive UV peak which was eluted between adenlne and the active peak from the Dowex 50 H+ columns was crystallized and indentified as 6-methylamino purine.