The isolation from human dentine and ox bone of phosphate-containing peptides.

Abstract

LEAVER, EASTOE and HARTLEY (1960) reported on the presence in dentine and bone of acid-soluble peptides rich in arginine which were believed to be associated with citric acid. Later LEAVER, SHU~TLEWORTH and TRIFFIT-~ (1965) separated bone citric acid from acid-soluble nitrogenous material which appeared to be a mixture of soluble collagen and various peptides. Though there has been no other interest in peptides in bone and dentine, those in enamel have recently been extensively studied by GLIMCHER and LEVINE (1966) who found that the organic matrix of bovine enamel was mainly composed of small peptides rich in glycine, aspartic acid, glutamic acid and serine, the latter often occurring as its phosphate ester. The present report describes a search for basic, phosphate-containing peptides in bone and dentine and is one aspect of an extensive investigation into the acid-soluble nitrogen of these tissues. Ox bone (100 g) and human dentine (70 g) were used in this investigation. The bone was stripped of all adhering tissues, allowed to stand in cold ethanol, air dried and split into chips in a percussion mortar. The roots of freshly extracted teeth were cut off with a diamond disc, the pulp chamber being ground out with a dental bur which was also used to remove cementum. After standing in cold ethanol, the dentine was dried in air. The further treatment of bone and dentine was identical. The tissue was treated with sufficient N HCl at 2°C to achieve demineralization. Insoluble material was filtered off and the pH of the filtrate adjusted to 5.0 with sodium bicarbonate before the addition of a slight excess of staurated potassium oxalate solution. The precipitated calcium oxalate was centrifuged off and the supernatant rotary evaporated to dryness at 37°C. The solids were then taken up in 05 N HCl and applied to a column of Amberlite IR-120 cationic exchange resin. The U.V. absorption of the eluate at 280 rnp was monitored. Continued elution with O-5 N HCI produced a fraction (A) which contained organic acids, phosphoric acid and some acid-reacting nitrogenous material. After washing with water, which removed minimal amounts of nitrogenous material, elution with N NH,OH was used to give a fraction (B) rich in nitrogen and free from salts and organic acids. This fraction was then applied to a Sephadex G-25 column, 6-ml fractions being collected and monitored as previously. A fraction (Bl), appearing immediately after 1209