Chromatography on calcium hydroxylapatite of glycoproteins solubilized by triton X-100 from particulates prepared from rat brain

Abstract

Aqueous solvents are capable of extracting only 10% of the glycoproteins of the combined mitochondrial and microsomal fractions prepared from rat brain. Solutions that contain 0.5% (v/v) Triton X-100 extract 60% of the glycoproteins. Triton-solubilized glycoproteins can be fractionated by chromatography on calcium hydroxylapatite columns with 100% recovery, although considerable cleavage of the protein-bound N-acetylneuraminic acid occurs during the procedure. Approximately 20% of the applied glycoproteins are not adsorbed to the gel and are recovered with a 2.3-fold purification. A large part of the glycoproteins that adsorb to the gel fail to adsorb upon rechromatography and are recovered with a 2- to 3-fold purification. Glycoproteins present in whole rat brain tissue or tissue extracts were assayed by determining the amount of N-acetylneuraminic acid and hexosamine present in the glycopeptides released by the proteolytic action of papain on the defatted protein residue that remains after extraction of the sample with chloroform-methanol (2:1, v/v). Dialyzable glycopeptides (DGP) and nondialyzable glycopeptides (sialofucohexosaminoglycans, SFHG) are released by proteolysis. The ratio of SFHG/DGP recovered from the glycoproteins present in various extracts and purified fractions prepared from these extracts remains constant, suggesting that both of these glycopeptides are derived from the same glycoprotein or glycoproteins.