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Calibration of the relative molecular mass of proteoglycan subunit by column chromatography on Sepharose CL-2B

1986; Portland Press; Volume: 235; Issue: 2 Linguagem: Inglês

10.1042/bj2350553

1470-8728

Hiroyuki Ohno, John Blackwell, A. M. Jamieson, David A. Carrino, Arnold I. Caplan,

Polysaccharides and Plant Cell Walls

Calibration relationships were derived for cartilage proteoglycan subunit (PGS) that relate the inverse z-average hydrodynamic radius (Rs) and the weight-average Mr (Mw) to the partition coefficient (Kav.) for PGS when chromatographed on a Sepharose CL-2B column. PGS isolated from chick limb-bud chondrocyte cell cultures was fractionated chromatographically into eight pools, for which Mw and Rs were determined by total-intensity and dynamic light-scattering measurements. These data were found to be related to Kav. through the following empirical equations: log Mw = -(1.65 +/- 0.27)Kav. +(6.58 +/- 0.08); log Rs = -(0.69 +/- 0.04)Kav. +(2.75 +/- 0.01). Application of these relationships to the chromatographic data led to Mw = 1.48 × 10(6) and Rs = 38.7 nm (387 A) for the unfractionated specimens compared with values of Mw = 1.46 × 10(6) and Rs = 38.2 nm (382 A) determined by light-scattering. Our results were found to be consistent with previously proposed phenomenological models for the gel-filtration mechanism. Application of these calibration relationships to Kav. for several unfractionated specimens led to predicted values of Mw and Rs that are accurate to within 10%.