The effect of mucopolysaccharide linkage region and intrachain alkali labilities on chain weight determination.

The validity of the reducing end group (copper reduction) method for determination of chain weight of chondroitin sulfate is shown by comparison with chain weight determination from linkage region components, xylose and galactose. It is established that chains of both chondroitin sulfate and keratan sulfate are stable to alkali as strong as 2.0 N NaOH at room temperature for at least 120 hours. The existence of two populations of protein linkage regions in both chondroitin sulfate and keratan sulfate from both the “light” and “heavy” fractions of proteoglycan from human nucleus pulposus is shown. These linkage regions are classified as alkali-labile, which is split by 0.2 N NaOH at room temperature, and alkalistable, which is stable to 0.2 N NaOH at room temperature but which is readily split by 2.0 N NaOH at room temperature. Sixty-two per cent of the linkage regions of both chondroitin sulfate and keratan sulfate in the light fraction of proteoglycan were alkali-labile, while 78% of linkage regions of both mucopolysaccharides in the heavy fraction were alkali-labile. It is pointed out that the individual chain weights of the alkali-labile and the alkali-stable fractions cannot be determined from reducing end group determinations alone. The relative molecular sizes and amounts of chondroitin sulfate and keratan sulfate bound by alkali-labile and alkalistable linkages in both light and heavy fractions of proteoglycan are investigated by gel filtration. Removal of mucopolysaccharide from protein by 2.0 N NaOH allows the separation by gel filtration of the protein of proteoglycan into two peaks differing in amino acid composition and molecular size. It is observed for the chondroitin sulfate studied that reducing end group determination after 0.2 N NaOH treatment should give fairly reliable values for the chain weight of the alkali-labile fraction.