Masashi Kosakai

A partial modification of the carbazole method of Bitter and Muir for quantitation of hexuronic acids.

Abstract The carbazole method of T. Bitter and H. M. Muir (1962, Anal. Biochem. 4 , 330–334)for quantitation of hexuronic acids was modified partially. In this modification (procedure g), 0.2 m anhydrous sodium tetraborate was used instead of 0.025 m sodium tetraborate decahydrate. The color intensity of d -glucuronolactone was slightly diminished by this modification, however, that of l -iduronolactone significantly increased. Thus, the difference in color intensity between d -glucuronolactone and l -iduronolactone became very small. When evaluated by procedure g, the hexuronic acid content of dermatan sulfate was comparable to that of chondroitin sulfate A.

A rapid method for separation and identification of several hexuronic acids and hexuronic acid-containing oligosaccharides

Abstract Five naturally occurring hexuronic acids and several hexuronic acid-containing oligosaccharides were separated and identified by high voltage paper electrophoresis, using one of the following buffers. (i) Pyridine-acetic acid-water (1:10:89, by volume), the pH of which was adjusted to 2.3–3.5 with 98% formic acid. (ii) Pyridine-water (1:90, by volume), the pH of which was adjusted to 3.5–4.0 with glacial acetic acid. The best separation of the five hexuronic acids and heparin disaccharides was observed at pH 2.7 after electrophoresis for 180 min at 100 V/cm. At pH 3 l -gulosyluronic acid- l -guluronic acid could be easily isolated from an acid hydrolysate of alginate by the present method.

Characterization of fibronectin from fetal human plasma in comparison with adult plasma fibronectin

Fibronectin was purified from fetal human plasma and characterized in comparison with fibronectin from adult human plasma. Fetal plasma fibronectin had an amino-acid composition, immunological properties, and cell-attachment-promoting activity similar to those of adult plasma fibronectin. However, fetal plasma fibronectin was shown to have a distinct carbohydrate composition which is characterized by the presence of fucose. To ascertain the differences in the carbohydrate moiety, 14C-labeled glycopeptides were prepared and sequentially analyzed with columns of immobilized concanavalin A and lentil lectin. Glycopeptides from fetal plasma fibronectin contained a population of glycopeptides which bound to both lectin gels. Almost all of the glycopeptides in this population lost their ability to bind to lentil lectin upon fucosidase digestion, indicating that fetal plasma fibronectin possesses a substantial amount of fucosylated biantennary glycans. In contrast, glycopeptides from adult plasma fibronectin practically lacked such glycopeptides. Another difference observed was that fetal plasma fibronectin had a larger amount of concanavalin A-unbound glycopeptides than did adult plasma fibronectin. These results indicate the presence of age-related variation in glycosylation of human plasma fibronectin.

Epimerization of D-glucuronic acid to L-iduronic acid by deamination of heparins

Abstract Porcine α-heparin, whale ω-heparin and 4-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-glucuronic acid were deaminated with nitrous acid. Uronic acid in the deamination products was identified as L-iduronic acid by paper chromatography, gas-liquid chromatography, infrared spectrophotometry and carbazole-to-orcinol ratio of the color reaction. The present data indicate that the deaminative cleavage of the glycosidic linkage between C4 of D-glucuronic acid and C1 of D-glucosamine results in the concomitant epimerization of D-glucuronic acid to L-iduronic acid.

Isolation and characterization of heparin disaccharides.

Abstract Purified porcine heparin was hydrolyzed with 0.5m hydrochloric acid for 20 h at 80°. The neutralized hydrolyzate was subjected to gel filtration through Sephadex G-10. Fractions containing products of higher molecular weight than disaccharide were treated repeatedly by the foregoing procedure. The disaccharide-containing fractions thus obtained were purified by preparative, high-voltage paper electrophoresis at pH 1.9, two disaccharides were then separated by multiple, preparative paper-chromatography. The yields or the disaccharides (P-4 and P-5) were 13.1 and 3.8 mg, respectively, in terms of D -glucuronic acid, from 300 mg of the starting heparin. As P-4 was not homogeneous, it was further purified by preparative, high-voltage paper electrophoresis at pH 2.7. Analytical data of P-4 and P-5 of their N -acetyl derivatives (P-4A and P-5A) before and after reduction with sodium borohydride indicated that P-4 and P-5 consisted of equimolar amounts 2-amino-2-deoxy- D -glucose and hexuronic acid, having hexuronic acid at the reducing end. The hexuronic acid in P-4A and P-5A was determined by g.l.c. to be L -iduronic acid and D -glucuronic acid, respectively. After reduction of the products obtained by Smith degradation of the reduce P-4A and P-5A, threitol and erythritol, respectively, and also glycerol were detected by g.l.c. The α-anomeric configuration of the 2-acetamido-2-deoxy- D -glucopyranosyl linkages of P-4A and P-5A was suggested by i r. and p.m.r. spectra and by the high dextrorotations; ( values of P-4A and P-5A were +91.6° (in water) and + 114.9° (in water), respectively. The foregoing observations indicated that P-4 and P-5 were O -(2-amino-2-deoxy-α- D -glucopyranosyl-(1→4)- L -idopyranuronic acid and O -(2-amino-2-deoxy-α- D -glucopyranosyl)-(1→4)- D -glucopyranuronic acid, respectively.

Isolation and characterization of a mucin-type glycoprotein from the lung lavage of a patient with alveolar proteinosis

A high molecular weight glycoprotein was isolated from the lavage fluid of a patient with alveolar proteinosis by gel chromatography with Sepharose CL-4B. The glycoprotein gave a single band stainable with alcian blue and with periodate-Schiff reagent on the cellulose acetate membrane electrophoresis. The glycoprotein did not penetrate 3.3% polyacrylamide gel but moved into 1% agarose gel as a periodate-Schiff positive single band, when electrophoresed in the presence of sodium dodecyl sulfate. The chemical analysis and the results of the beta-elimination reaction showed the presence of O-linked carbohydrate chains characteristic for a mucin-type glycoprotein. These data provide the first characterization of a mucin-type glycoprotein isolated from lung in pulmonary alveolar proteinosis.