Heparin enzymic analysis

The heparin degradation rate at any radial position inside the catalyst particle is proportional to the bound heparinase concentration at that position. If the immobilized enzyme concentration is not uniform, the conventional analysis of simultaneous diffusion and reaction within a porous catalytic particle must be modified. The reaction rate within the catalyst particle will have an explicit radial dependence introduced via the enzyme concentration, as well as a dependence on the substrate concentration. 

Preparation of the sample. Either semm or plasma (oxalated or heparinized) may be used for the determination. This may be freshly drawn blood from an animal. (Do not do this yourself your instructor will supply the sample.) See Chapter 1 for a discussion of the differences between semm, plasma, and whole blood. A 10- to 15-mL sample (20 to 30 mL whole blood) should be adequate for triplicate determinations by a class of 30 students. Fluoride should be added to prevent glycolysis, or breakdown of glucose, which can change the pH. The fluoride inhibits the enzyme catalysis causing glycolysis and stabilizes the pH for about 2 h. The tube used for collecting the sample can be rinsed with a solution of 100 mg sodium heparin plus 4 g sodium fluoride per 100 mL. The sample should be kept anaerobically, that is, stoppered to keep out atmospheric CO2. Since the analysis should be done on the day the blood is drawn, the solutions should be prepared ahead of time. 

Blood Venous blood samples are routinely collected for different laboratory tests and are suitable for molecular analysis. Blood contains a number of enzyme inhibitors that can interfere with downstream DNA analysis. In addition, common used anticoagulants such as heparin and EDTA can interfere with downstream assays as a consequence the DNA isolation from blood requires a standardized method to provide high-quality DNA without contaminants or enzyme inhibitors. 

The h.p.l.c. analysis of unsaturated disaccharides obtained by enzymic hydrolysis of heparan sulphate and heparin was found to be more rapid and much more sensitive than the previously described method which employed paper chromatography. ... 

Galactosamine, glucosamine and 26 amino-acids have been analysed by h.p.l.c. as their N-phenylthiocarbamate derivatives. Unsaturated disaccharides generated by enzymic cleavage of heparin sulphates have been separated on a polystyrene-based cation exchange resin, while unsaturated disaccharides with 0-3 sulphate moieties similarly released from chondroitin sulphate, have been separated on a primary amine gel, with post-column reaction with 2-cyanoacetamide and fluorimetric detection. The reversed-phase analysis of l-N-( -D-glucopyranosyl)amobarbital diastereomers in urine has also been reported. ... 

High pressure capillary electrophoresis was used in the determination of mono-, di- and tri-sulfated unsaturated disaccharides released enzymically from glycos-aminoglycans. In related work, capillary electrophoresis and polyacrylamide gel electrophoresis were applied to the analysis of low molecular weight heparins and heparin oligosaccharides, and the former method was used to monitor selective pivaloylation of a trisulfated unsaturated disaccharide derived from heparin. ... 

Oligosaccharides (mostly DP 2-6) released by various enzymic digestions of hyaluronic add were derivatized with l-(4-methoxyphenyl)-3-methyl-5-pyrazolone (to give u.v.-absotbingderivatives of an uncharacterized structure) for reversed-phase h.p.lc. analysis. Oligosaccharides from the enzymic digestion of chondroitin sulfate and heparin were examined by ion-pair reversed-phase h.p.l.c. - electrospray m.s. analyses. ... 

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