The isolation of a protein containing manganese from the aleurone grains of the seeds of Arachis hypogaea L

Abstract

Five classes of subcellular particles (aleurone grains, protein bodies, starch grains, cell walls and reticulum) were isolated by a nonaqueous differential centrifugation technique and their manganese, copper and potassium contents were determined by nondestructive thermal neutron activation analysis. The aleurone grains have been shown to be the major depository for manganese, copper and potassium where the manganese and copper in the aleurone grains was found largely nondialyzable and associated with proteins while potassium is dialyzable. Conarachin, a seed protein fraction which contains the highest concentration of manganese and copper, was used as the starting material for the isolation of the manganoprotein which was assigned the trivial name of manganin. The isolation scheme for manganin was based on ammonium sulfate fractionation, a salt concentration reduction technique, differential precipitation at various pH values and gel filtration. Based on metal content, the minimum molecular weight of two preparations of manganin containing 982 μg and 971 μg of manganese per g respectively was calculated to be 55,900 and 56,600. The molecular weight as determined by ultracentrifugation according to the methods of Yphantis and Ehrenberg was found to be 57,900 and 56,800 respectively with sedimentation velocity analysis showing only one component while equilibrium sedimentation analysis showed a trace amount of low molecular weight material. Zone electrophoresis of manganin showed one migrating band at pH values of 7.6 and 5.0. The amino acid composition of manganin was determined and large amounts of aspartic acid, glutamic acid and glycine were found. Manganin appears to be a protein of about 57,400 molecular weight which contains one atom of manganese per molecule of protein.