The suggestion is made that, in solution, the flexible-chain molecules of dextran can undergo an osmotic compression as concentration is increased. Approaches are developed described the molecular shrinkage (i) as arising from intra- and inter-molecular forces, (ii) based on the molecular characteristics of the dextran, and (iii) as estimated by viscosity measurements. Comparison with the macroscopic shrinkage of cross-linked dextran (Sephadex) beads [Edmond, Farquhar, Dunstone & Ogston (1968) Biochem. J. 108, 755-763] is made. In all systems studied, the experimental estimates of compression, both from gel-shrinkage and viscosity measurements were in reasonable agreement with theoretical predictions. The interpretation of the viscosity concentration-dependence was applied to compact structures (albumin and Percoll). Their behaviour was in marked contrast with that of dextran. It is noted that molecular compression may be important in considering transport processes in and thermodynamic properties of concentrated systems.
A model connective-tissue system was developed that is amenable to the determination of permeability coefficients of diffusing solutes. The system involves the culture of 13-day chick-embryo chondrocytes on a Millipore filter (HA:0.45 micron pore size) to form what is, in effect, a confluent, extremely thin cartilage slice of uniform thickness. These cultured chondrocyte membranes were used to measure the steady-state flux of radioactively labelled low-molecular-weight solutes and micro-ions. Similarly, the permeability coefficients of either radioactively labelled or enzymically active proteins across the membranes were determined. The membrane was found to have no marked effects on the diffusional behaviour of low-molecular-weight non-electrolytes (water, proline, ribose, glucose, sorbitol, raffinose). For micro-ions (Na+, SO42-, Cl-, glutamate, glucuronate,), the diffusive behaviour was found to be markedly affected by the ionic strength of the solvent used in a manner which was consistent with a Donnan distribution resulting from the immobilized proteoglycans. Globular proteins permeated the membrane at rates which decreased as the molecular size of the diffusing solute increased. The apparent diffusion rates of fibrinogen and of collagen through the membranes were greater than would be expected on the basis of their diffusion coefficients in free solution. Reasons for this behaviour are discussed.
1. A bilayer strip, cut from a thin layer of cross-linked polyacrylamide gel cast on to cellulose tissue, forms an open circular loop whose ends are close together. Shrinkage of the gel, in response to the osmotic pressure of a non-penetrating solution, causes a proportional separation of the ends of the loop. This is measured with a microscope and micrometer eyepiece. 2. The resulting effective sensitivity is about 30 times that of the Sephadex-bead osmometer (Ogston & Wells, 1970), i.e. of the order of 5Pa, comparable with that of a membrane osmometer. Use of gel up to 70% (w/v) allows the measurement of molecular weights, as low as 1500 in favourable cases, with an accuracy of 1–2%. The useful range of osmotic pressure is up to 5kPa. A single measurement requires 0.5ml of solution. Equilibration is completed in 20–30min. 3. The method is illustrated by measurements on human serum albumin, ovalbumin, cytochrome c , samples of dextrans, polyvinyl alcohol, and polyethylene glycols 6000 and 1000.
1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca 2+ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro . 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p -chloromercuribenzoate or Cu 2+ ions.
1. Several protein–polysaccharides were isolated from the soluble extracts of bovine heart valves by sedimentation equilibrium in a caesium chloride density gradient (Meyer, Preston & Lowther, 1969). 2. Compositional and structural studies indicated that the polysaccharide moiety was chondroitin sulphate. Differences in the protein content of the products were observed. There was no evidence suggesting the presence of keratan sulphate. 3. Sedimentation studies indicated that the molecular weights of the samples were between 4.2×10 4 and 6.5×10 4 . The results are discussed in terms of a basic model for the protein–polysaccharides of two polysaccharide chains linked by a protein of variable size.
1. A soluble extract of bovine heart valves was obtained after the tissue had been pulverized at liquid-nitrogen temperatures in a mill. 2. Hyaluronic acid was isolated from the crude extract by sedimentation equilibrium in a caesium chloride density gradient (Franek & Dunstone, 1966). 3. Analysis of the product indicated that it contained 15% of protein and the molar ratio of glucuronic acid to glucosamine was 1·27. 4. Its physicochemical properties, as determined by lightscattering, viscosity and sedimentation studies, suggested that its molecular size and configuration were similar to those of hyaluronic acid isolated from ox synovial fluid (Preston, Davies & Ogston, 1965).