FAST MOVING AND SLOW MOVING HEPARINS, DERMATAN SULFATE, AND CHONDROITIN SULFATE - QUALITATIVE AND QUANTITATIVE-ANALYSIS BY AGAROSE-GEL ELECTROPHORESIS

被引:66
作者
VOLPI, N
机构
[1] Department of Biologia Animale, University of Modena, Modena
关键词
D O I
10.1016/0008-6215(93)84259-9
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Heparin from beef intestinal mucosa, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their structures and physico-chemical properties were evaluated by different techniques (disaccharide patterns by specific enzymatic cleavage, relative molecular mass by high-performance size-exclusion chromatography, sulfate-to-carboxyl ratio by potentiometric determination). Heparin was fractionated into ''slow moving'' and ''fast moving'' fractions by selective precipitation as the barium salt at different temperatures. The ''fast moving'' and ''slow moving'' components of heparin, dermatan sulfate, and chondroitin sulfate were utilized to run calibration curves in agarose-gel electrophoresis. Mixtures containing different amounts of these glycosaminoglycans were made and separated by agarose-gel electrophoresis, and these were analyzed quantitatively. For analysis of relative amounts, the area of each individual component of mixtures, obtained by photodensitometric readings, was divided by the sum of the areas of all glycosaminoglycans and expressed as a percentage. For analysis of absolute amounts, the area under the curve for each component of mixtures was fitted to specific calibration curves, and the amount of each glycosaminoglycan was calculated in mug. The quantitative procedure performed by analysing absolute amounts was used to obtain an accurate quantitative evaluation of each component in mixtures of glycosaminoglycans utilized for pharmaceutical purposes. A sensitive method was developed for the evaluation of very small amounts (0.2% w/w) of possible glycosaminoglycans as contaminants in preparations of a single species of glycosaminoglycan. This technique requires specific enzymatic degradation by bacterial lyases, separation in agarose-gel electrophoresis, and quantitative analysis by photodensitometric analysis and specific calibration curves.
引用
收藏
页码:263 / 278
页数:16
相关论文
共 37 条
[21]   HETEROGENEITY OF HEPARIN - CHARACTERIZATION OF 100 COMPONENTS WITH DIFFERENT ANTICOAGULANT ACTIVITIES BY A COMBINATION OF ELECTROPHORETIC AND AFFINITY-CHROMATOGRAPHY METHODS [J].
NADER, HB ;
TAKAHASHI, HK ;
GUIMARAES, JA ;
DIETRICH, CP ;
BIANCHINI, P ;
OZIMA, B .
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 1981, 3 (06) :356-360
[22]   MONITORING THE PURITY OF PHARMACEUTICAL HEPARIN PREPARATIONS BY HIGH-FIELD H-1-NUCLEAR MAGNETIC-RESONANCE SPECTROSCOPY [J].
NEVILLE, GA ;
MORI, F ;
HOLME, KR ;
PERLIN, AS .
JOURNAL OF PHARMACEUTICAL SCIENCES, 1989, 78 (02) :101-104
[23]  
OEGEMA TR, 1984, J BIOL CHEM, V259, P1720
[24]  
OGREN S, 1976, BIOCHEM J, V154, P605
[25]   STRUCTURAL DIFFERENCES OF DERMATAN SULFATES FROM DIFFERENT ORIGINS [J].
POBLACION, CA ;
MICHELACCI, YM .
CARBOHYDRATE RESEARCH, 1986, 147 (01) :87-100
[26]   EXAMINATION OF A POSSIBLE ROLE FOR DERMATAN SULFATE IN THE AGGREGATION OF COMMERCIAL HEPARIN SAMPLES [J].
RACEY, TJ ;
ROCHON, P ;
MORI, F ;
NEVILLE, GA .
JOURNAL OF PHARMACEUTICAL SCIENCES, 1989, 78 (03) :214-218
[27]   FRACTIONATION OF HEPARIN-DERIVED OLIGOSACCHARIDES BY GRADIENT POLYACRYLAMIDE-GEL ELECTROPHORESIS [J].
RICE, KG ;
ROTTINK, MK ;
LINHARDT, RJ .
BIOCHEMICAL JOURNAL, 1987, 244 (03) :515-522
[28]  
ROBINSON HC, 1978, J BIOL CHEM, V253, P6687
[29]   SUPRAMOLECULAR ORGANIZATION OF EXTRACELLULAR-MATRIX GLYCOSAMINOGLYCANS, INVITRO AND IN THE TISSUES [J].
SCOTT, JE .
FASEB JOURNAL, 1992, 6 (09) :2639-2645
[30]  
SMITH RL, 1980, ANAL BIOCHEM, V103, P191