PURIFICATION AND PROPERTIES OF ALPHA-GALACTOSIDASE OF GERMINATION VICIA SATIVA SEEDS

α‐Galactosidase from germinating Vicia sativa seeds has been extracted with a 0.9% NaCl solution. Treatment with manganous sulphate eliminated inactive fractions. The enzyme was precipitated from the supernatant with (NH4)2SO4 at 80% saturation, redissolved in water and dialyzed. Purification was further increased by three successive steps of column chromatography. Only fractions showing α‐galactosidase activity have been used for each subsequent step. Successive passage through two hydroxylapatite and one Sephadex column gave an enzyme with a specific activity 1,100 times higher than that of the crude extract, and with a yield of 10% from the original material. The enzyme thus obtained behaves as a single protein in electrophoresis on Cellogel. Sedimentation in a sucrose gradient and ultracentrifugation in an analytical ultracentrifuge suggest a molecular weight of about 30,000. The protein is rich in aspartic and glutamic acids and has a low cysteine content. Dansylation and the dinitrofluorobenzene method showed the presence of a single N‐terminal residue which was identified as l‐alanine. The purified α‐galactosidase seems therefore to be a single polypeptide chain. The pH optimum of the α‐galactosidase is 6.3. The enzyme is not very sensitive to thermal inactivation and is devoid of activity on α‐glucosides and β‐galactosides. It hydrolyses phenyl‐α‐galactoside rapidly and galactinol very slowly. Unlike the α‐galactosidase from coffee beans it does not liberate galactose from the galactomannans of Leguminoseae. Michaelis constants at pH6.3 are markedly different for the different substrates: Km= 9.7 × 10−4 for p‐nitrophenyl‐α‐galactoside; 3.8 × 10−3 for phenyl‐α‐galactoside; 5.8 × 10−2 for raffinose and 4.1 × 10−1 for galactinol. In the case of the two phenol galactosides a strong inhibition was observed with excess substrate. This inhibition does not take place with raffinose or galactinol as substrate. α‐Galactosidase is inhibited by Hg2+, Ag+ and weakly activated by Fe3+, Zn2+ and Mn2+. A transferase activity was observed with phenyl‐α‐galactoside as donor and various acceptors: glucose, mannose, galactose, cellobiose and sucrose. Raffinose also acts as an α‐galactosyl donor. With galactinol as acceptor the following compound is formed: Galpα1‐6Galpα‐1′‐myoinositol. Copyright © 1969, Wiley Blackwell. All rights reserved