A crystalline glycopeptide from normal human urine.

There is abundant evidence that glycosaminoglyeans are linked covalently to the protein in tissues (Mathews & Lozaityte, 1958; Muir, 1958). This fact suggested that isolation and characterization of glycosaminoglycan-peptides, particularly uronic acid-containing glycopeptides in normal urine, will assist in the understanding of the normal metabolism of glycosaminoglycans in tissues. The present study was undertaken to isolate and characterize glycopeptides containing uronic acid in normal human urine. Hakomori, Kawanchi & Ishimoda (1962) isolated three glycopeptides from urine, but none contained any uronic acid. One of them resembled a glycopeptide isolated from aorta connective tissue. Cherian & Radhakrishnan (1966) have isolated from human urine a uronic acidcontaining glycopeptide, the amino acid composition ofwhich suggested that it was derived from collagen. Materials and methods. Sephadex G-50, Sephadex G-25 and DEAE-Sephadex A-50 were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Dowex 1 (X8; 200-400 mesh) resin was purchased from J. T. Baker Chemical Co., Phillipsburg, N.J., U.S.A. A.R.-grade reagents were used throughout this study and all organic solvents were redistilled before use. Combined urine from male laboratory workers (24-40 years old) was collected with toluene and chloroform as preservatives. The urine was stored at 40 (not more than 48 hr.) until processing and was then concentrated at 400 under reduced pressure to one-fifth of its original volume. The concentrated urine was dialysed at 40 against running tap water for 24 hr. Any precipitate formed was discarded. The dialysed supernatant was treated with alkaline cetyltrimethylammonium bromide and potassium thiocyanate solution. The precipitate formed during the treatment was discarded. The treated urine supernatant was concentrated at 400 under reduced pressure to oneeightieth of its original volume. The concentrated urine supernatant was dialysed at 40 against four changes of deionized distilled water for 20 hr. The non-diffusible material of the dialysis bag was further concentrated at 400 under reduced pressure to one-hundredth of the original volume and was chromatographed on a Sephadex G-50 column (53 cm. x 4-5 cm.). The column was eluted with 01M-NaCl. The fractions eluted between 750 and 1020ml. were pooled and concentrated at 400 under reduced pressure to one-fifth of the volume. The concentrated material was then chromatographed