Purification and Properties of Chlorophyllase from Greened Rye Seedlings1

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Abstract
1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1 % cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of frac-tionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39, 000. 3. The purified enzyme was stable at pH 6–9 and 4°C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30°C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodo-spirillum rubruminto bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacterio-chlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30°C in the presence of 30% acetone, the Km values and specific activities were 12 μM and 480 nmol.min−1.mg−1 for chlorophylls a and 4 μm and 170 nmol.min−1.mg−1 for R. rubrum bacteriochlorophyll a, respectively.