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The exclusion of large solutes by cartilage proteinpolysaccharide
Author(s) -
Gerber Bernard R.,
Schubert Maxwell
Publication year - 1964
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.1964.360020307
Subject(s) - chemistry , chondroitin sulfate , salt (chemistry) , albumin , urea , macromolecule , chromatography , serum albumin , bovine serum albumin , chondroitin , biochemistry , glycosaminoglycan , organic chemistry
The dialysis technique of Ogston and Phelps, using a Millipore filter as a membrane to measure the effect of a nondialyzable macromolecule on the distribution of dialyzable macromolecules, has been used to measure the effect of a compound of protein and polysaccharide (PP‐L), isolated from cartilage, on the distribution of some dialyzable macromolecules. PP‐L (1%) in buffer acts as if part of its solution volume were unavailable as follows: 55% for serum albumin, 64% for chondroitin sulfate made by alkaline degradation of PP‐L, and 98% for chondroitin sulfate made by trypsin degradation of PP‐L. The use of different buffers, variation of pH from 5 to 10, variation of temperature from 5 to 42°C., variation of salt concentration from 0.15 to 2 N , and variation in ethanol concentration from 0 to 30% had no effect on the distribution of albumin, which was always excluded to about the same extent from the PP‐L solution. This effect could account for the virtual absence of serum proteins and of free chondroitin sulfate from cartilage. At low salt concentration (0.10–0.02 N ) and at 5°C, the distribution was reversed and albumin behaved as if strongly bound by PP‐L. At low‐levels of albumin this binding of PP‐L increased linearly with albumin concentration. This binding was partly reversed by raising the temperature or by adding urea, and completely reversed by the addition of salt. Polystyrene sulfonate, a nondiffusible polyanion, had an effect similar to that of PP‐L in excluding chondroitin sulfate from its solutions. It showed a very strong binding effect for albumin which persisted even in the presence of salt as high as 2 N . A quantitative comparison has been made between the excluded volume effect of the soluble macromolecules PP‐L and hyaluronate and the effect of insoluble Sephadex particles to exclude large molecules from their interiors. The technique of Ogston and Phelps is thus valuable‐ not only for measuring the exclusion of diffusible macromolecules from part of the solution volume of nondiffusible macromolecules, but also for measuring the existence and extent of binding of diffusible macromolecules to nondiffusible macromolecules.