Quantitative Isolation of Purified Acidic Glycosaminoglycans From Rat Skin

Abstract

A simple method is proposed for the quantitative isolation of purified glycosaminoglycans from 1- to 3-g samples of rat skin. The cetylpyridinium complex of the polysaccharide is isolated from a clarified papain digest of fresh skin. After recovery of the glycosaminoglycan from the complex, residual protein is removed by treatment, in sequence, with sodium hydroxide, phenol, and trichloroacetic acid. The product contains only small amounts of non-hexosamine nitrogen.The optimal conditions for each step of the procedure were developed by systematic experimentation. The apparent loss of carbazole–borosulfuric chromogen in the formation of cetylpyridinium chloride complex (CPC) seems unlikely to be due to glycosaminoglycan; no other significant losses occurred. The recovery of added glycosaminoglycans was close to theory. The method gave results agreeing well with those for standard published procedures. Trial applications revealed the method to be barely precise enough to distinguish between animals.

Degeneration and the chemical composition of the human lumbar intervertebral disc

“…These values agreed well with other analyses of collagen (8). A second portion of freeze-dried tissue was suspended in 0.1 M Na acetate buffer, pH 5.5, containing 5 mM Na, EDTA at 60°C, and then 0.3 mg activated papain (PAP, Worthington Biochemical, Freehold, NJ, U.S.A.) and L-cysteine to 5 mM were added and the mixture was digested overnight (16). The digest was centrifuged to remove a trace of insoluble residue, and the supernatant was analyzed for hexuronate (3).…”

The response of the canine intervertebral disc to immobilization produced by spinal arthrodesis is dependent on constitutional factors

“…These were filtered through a cotton wool plug to remove particulate matter. Residues were washed thoroughly with distilled water, lyophilized, and analyzed for hexuronic acid content after papain digestion (38). The total hexuronic content of the tissue was estimated as the sum of that present in extracts and residue.…”

The Matrix Components of the Epiphyseal Growth Plate and Articular Cartilages From Dogs Treated With Ammonium Tetrathiomolybdate, a Copper Antagonist

“…Metabolic studies and proteoglycan isolation were undertaken using fresh tissues, all other studies were conducted on lyophilised samples. Hexuronic acid content was determined by the method of Blumenkrantz and Asboe-Hansen (1973) or Farndale, Sayers and Barrett (1982), after papain digestion {Pearce and Mathieson, 1967). Protein was determined using the Peterson (1977) modification of the Lowry procedure.…”

“…The full thickness of growth plate tissue was harvested from both the femoral and tibial growth plates, pooled and diced in sterile icecold 0-9% saline and set up in organ culture as described previously (McKenzie et al, 1977) but with the following modification. Tissue samples were solubilised by overnight digestion at 60° with crystalline papain 0-5 mg/ml in 0-05 M phosphate buffer pH 7-0; containing 0-01 M EDTA and 0-01 M cysteine (Pearce and Mathieson, 1967). Glycosaminoglycans (GAGs) were then precipitated with ice-cold absolute ethanol and the precipitate dried in a vacuum desiccator.…”