Determination of sulphate/uronic acid ratios in glycosaminoglycans separated by electrophoresis on cellulose acetate

Abstract

Glycosaminoglycans fractionated by electrophoresis on cellulose acetate strips which were stained with toluidine blue and scanned in wet condition showed optical density spectra with an absorbance maximum at 525 nm and a second maximum at 595–610 nm. The maximum at 525 nm was found to depend on the presence of sulphate groups. The maximum at 595–610 nm was present in all glycosaminoglycans investigated including hyaluronic acid and keratan sulphate. The ratio between the integrals of absorbance at 525 and 645 nm of the electrophoretic fractions showed a linear relationship with the sulphate/uronic acid ratio. This was true for a uronic acid range from 1.75 to 6.25 nmol and a sulphate/uronic acid range from 0.34 to 1.72 mol/mol. The pH value of the electrophoresis buffer and the presence of protein in the sample had no influence on the absorbance ratio. Hence preincubation with chondroitinases could be done. Keratan sulphate on the other hand could not be studied by the method because the absorbance ratio of this glycosaminoglycan was too small. In repeated determinations on urinary and dermal glycosaminoglycans the method usually resulted in coefficients of variation of 5 to 15%.

Key Words:

• chondroitin 4 sulphate
• dermatan sulphate
• heparan sulphate
• hyaluronic acid
• keratan sulphate
• toluidine blue