STUDIES ON HOMOGENTISICASE*

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Abstract
The Fe++-bound and Fe++-free enzyme preparations were isolated from acetone-dried powder of beef liver, extracted at pH 9 precipitated by adding NaCl (final concentration, 0.6%) and 0.5 volume acetone. The material was fractionated in (NH4)2 SO4 (0.45-0.80 saturation), adsorbed on Ca3 (PO4)2 gel, and dialyzed against 0.01 [image] phosphate buffer, pH 7.0 (Fe++-bound enzyme) or 0.01 [image] acetate buffer, pH 5.0 (Fe++-free enzyme). The dialyzed preparation was purified about 50-fold. Rebinding of Fe++ to the Fe++-free enzyme took place rapidly in neutral and alkaline solutions, but very slowly in acid solutions. The exchange of exogenous Fe59 proceeded only when the enzyme was acting on the substrate. Any appreciable amount of Fe+++ was not detectable during the enzymic reaction.