Chondroitin 4-sulfate, chondroihin 6-sulfate, and darma

malography on a column of Con A-Sapharosa (Pharmacia Fine Chemicals Inc., Piscahaway, N. J.). The CEA sample was dissolved in Con A buffer (1.0 M NaCi in 0.1 M sodium acetate buffer, pH 6.0; 1 mM in each of CaCI2, MgCi2, and MnCl2) and applied to a waler-jacketed column (22 x 1.5 cm) of Con A-Sepharose. Pool 1 material was obtained by alul ing the column at 4°with 1.5 column volumes of Con A buffer. Pool 2 material wassubsequentlyeluhedat 37° using 3 column volumes of 20% (w/v) a-methyl-D-mannopymano side, Grade Ill (Sigma Chemical Co., St. Louis, Mo.) in ConA buffer. Both pools weredialyzedexhaustivelyagainst distilled waler and lyophilized. Radioimmunoassayswere performed using the triple-iso hope,double-antibody procedure described previously (4). Chondroitin 4-sulfate, chondroihin 6-sulfate, and darma tan sulfate were obtained from Miles Chemical Company, Division of Miles Laboratories, Inc., Elkhart, md. Hepanin (Grade 1), hyaluronic acid (Grade1), and bovine testicular hyalumonidasa(type I; about 300 NF units/mg) were ob hamadfrom Sigma. Haparansulfate was a gift from Dr. J .A. Cifonelli, Department of Pediatrics, University of Chicago, Chicago, Ill. Elactrophorasis of mucopolysacchanideswas carried out on cellulose acetate strips in 0.1 M formic acid-pynidina buffer, pH 3, according to the procedure of Hard (9). A Beckman Model R-10i Microzona Cell was used with mam branas manufactured for Beckman by Millipora Corp. , Bad ford, Mass.The mucopolysaccharidebandsware visualized by staining with 1% Alcian blue (Eastman Kodak Co., Roch ashen,N. V.) in 5% acetic acid. Mucopolysacchanides were also estimated using the rapid haxuronic acid assayprocedure of Bitter and Muir (1). Neutral and amino sugars ware analyzed using an im proved version4 of the procedure described by Etchison and Holland (5). Following methanolysis and ne-N-acahylation the samples ware trimathylsilylated using 0.1 ml of Tni-Sil “z,, (Pierce Chemical Co., Rockford, Ill.). Thesamples were transferred to silylation vials and placed in the automatic sample injector of a Hewlett-Packard Model 7620A gas chromalograph. A 6-ft glasscolumn (2 mm inside diameter) packed with 3.8% UCW-98on Gas Chrom 0, 100 to 120 mesh (Applied Science Laboratories, Inc., Stale College, Pa.),was used. Thecarrier gaswaschromatographic-grade nitrogen at a flow rate of 60 mI/mm. Peakswere automati cally integrated using an Autolab SystemIVcomputing inhe grator. Massspeclromatry of the trimethylsilyl methyl glycosidas