High-performance size-exclusion chromatography of porcine colonic mucins : Comparison of Bio-Gel ® TSK 40XL and Sepharose ® 4B columns

Abstract

A high-performance size-exclusion chromatography (HPSEC) method was developed for the separation of porcine colonic mucins using a Bio-Gel ® TSK 40XL HPSEC column (300 mn × 75 mm). In addition, porcine gastric and bovine submaxillary mucin preparations were used to describe more fully the separation characteristics of the HPSEC column. For comparison, the same preparations were also separated using a Sepharose ® 4B column (100 cm × 2.6 cm). The colonic and gastric mucins eluted in the void volume ( V 0) of both columns. Bovine submaxillary mucin was in the elution volume ( V e) of both columns. Analytical HPSEC of fractions ( V 0 and V e) of the various preparations obtained by Sepharose 4B chromatography exhibited retention times identical to those for fractions obtained by HPSEC. After separation by both methods, purified mucins were obtained by CsCl 2 density gradient ultracentrifugation; analytical HPSEC profiles, protein contents, and monosaccharide compositions of both gastric and colonic mucins from either column were similar. The HPSEC method, however, is ideally suited to separate microgram to milligram quantities of colonic mucin preparations quickly: 2 to 4 h, compared with 24 to 30 h for the Sepharose 4B method.