Enzymic methods

In 1993, the di-D-fructose dianhydrides were summarized as being of little, if any, commercial importance. 73 However, a search of the literature reveals an appreciable number of patents issued since 1989 for the manufacture of these compounds. These include enzymic methods for the production of individual dianhydrides (Ref. 130) or methods of production of mixtures using anhydrous HF or pyridinium poly(hydrogen fluoride) (see Ref. 131). Most cite the di-D-fructose dianhydrides as low-calorie sweetening agents (Ref. 132), and some claim anti-cariogenic properties (Refs. 132 and 133). 

Analytical Criteria for Clinical Enzyme Methods. The great variety of methods available for certain of the clinically useful enzymes can be confusing in the absence of criteria for assessing these methods. These criteria should be similar to those used to judge other quantitative clinical chemistry methods, and should also take into account the kinetic nature of the reaction. Some years ago, Amador and Wacker (17) suggested the following criteria, which are now widely us ... 

Clinical Criteria for Clinical Enzyme Methods. While it is possible to measure the activity of several thousand enzymes in body samples, in most cases it is practical to measure only a few while obtaining clinically useful results. In order to select the enzyme measurements which will be most useful, the following guidelines are suggested ... 

Kobos, R. K. Potentiometric Enzyme Methods, in Ion-Selective Electrodes in Analytical Chemistry, Vol. 2 (Freiser, H., ed.) New York Plenum Press, 1980, p. I... 

Tourn, M. L., Lombard, A., Belliardo, F., and Buffa, M. (1980). Quantitative analysis of carbohydrates and organic acids in honeydew, honey and royal jelly by enzymic methods. J. Apicult. Res. 19,144 146. 

All specimens were analyzed by EMIT (Enzyme Immunoassay Technique), an enzyme method based upon the competitive bonding of an enzyme and an antibody. This method yields a positive result with concentrations of 75 ng of PCP/ml or greater (Rubenstein et al. 

For detailed description and discussion of methods of separation and characterization of GAG, the reader is referred to specific mono-graphs38-42-46-47 dealing with the advantages and drawbacks of different colorimetric, titrimetric, electrophoretic, chromatographic, spectroscopic, and enzymic methods for the qualitative and quantitative characterization of heparin and its most common contaminants. The present article is concerned only with analytical aspects of relevance to the structural characterization of heparin. 

Isobe, K. and Nishise, H. (1995) A new enzymic method for glycolaldehyde production from ethylene glycol. Journal of Molecular Catalysis B-Enzymatic, 1 (1), 37—43. 

Many neurotransmitters are inactivated by a combination of enzymic and non-enzymic methods. The monoamines - dopamine, noradrenaline and serotonin (5-HT) - are actively transported back from the synaptic cleft into the cytoplasm of the presynaptic neuron. This process utilises specialised proteins called transporters, or carriers. The monoamine binds to the transporter and is then carried across the plasma membrane it is thus transported back into the cellular cytoplasm. A number of psychotropic drugs selectively or non-selectively inhibit this reuptake process. They compete with the monoamines for the available binding sites on the transporter, so slowing the removal of the neurotransmitter from the synaptic cleft. The overall result is prolonged stimulation of the receptor. The tricyclic antidepressant imipramine inhibits the transport of both noradrenaline and 5-HT. While the selective noradrenaline reuptake inhibitor reboxetine and the selective serotonin reuptake inhibitor fluoxetine block the noradrenaline transporter (NAT) and serotonin transporter (SERT), respectively. Cocaine non-selectively blocks both the NAT and dopamine transporter (DAT) whereas the smoking cessation facilitator and antidepressant bupropion is a more selective DAT inhibitor. 

Volkl K.P., Opitz N., Lubbers D.W., Continuous measurement of concentrations of alcohol using a fluorescence-photometric enzymic method, Fresenius J. Anal. Chem. 1980 301 162. 

The only example of this technique applied to the amylose component is that already described, of the action of Z-enzyme on the /3-limit dextrin. In the case of amylopectin, enzymic methods enable a distinction to be made between the proposed laminated and highly ramified structures (I and III, in Fig. 1, page 352). The method used by Peat and coworkers101 involves the successive action of /3-amylase and R-enzyme on waxy maize starch. /3-Amylolysis will degrade A-chains down to two or three units from the 6 —> 1-a-D interchain linkages. These latter linkages will protect the... 

B-chains until they are acted on by R-enzyme, when maltose or malto-triose will be produced from the residual A-chain, and linear dextrins from the B-chains. The amount of maltose or maltotriose liberated on treating the /3-limit dextrin with R-enzyme will be a measure of the number of A-chains in the molecule, and from these data, the ratio of A B chains in the molecule can be calculated.220 Peat concluded that multiple branching is an intrinsic part of the amylopectin structure, as the observed yield of these sugars was greater than expected for a singly-branched structure. It should be noted that glycogen has been shown by similar enzymic methods to possess a truly random structure.221... 

As a consequence of the previous considerations Kieber et al. [75] have developed an enzymic method to quantify formic acid in non-saline water samples at sub-micromolar concentrations. The method is based on the oxidation of formate by formate dehydrogenase with corresponding reduction of /3-nicotinamide adenine dinucleotide (j6-NAD+) to reduced -NAD+(/3-NADH) jS-NADH is quantified by reversed-phase high performance liquid chromatography with fluorimetric detection. An important feature of this method is that the enzymic reaction occurs directly in aqueous media, even seawater, and does not require sample pre-treatment other than simple filtration. The reaction proceeds at room temperature at a slightly alkaline pH (7.5-8.5), and is specific for formate with a detection limit of 0.5 im (SIN = 4) for a 200 xl injection. The precision of the method was 4.6% relative standard deviation (n = 6) for a 0.6 xM standard addition of formate to Sargasso seawater. Average re-... 

Enzymic methods are usually very specific and sensitive. Unfortunately the only methods in the literature for carbohydrates are all for glucose. Hicks and Carey [163] reported such a method, with a fluorometric final measurement, which was down to 3 x 10 8 M. Andrews and Williams [ 164] used a preconcentration step, sorption onto charcoal, elution, and a final determination with glucose oxidase. 

NADH, containing a tertiary amine functional group, has been readily determined by Ru(bpy)32+ ECL. However the oxidized form, NAD+, containing an aromatic secondary amine group produces virtually no ECL signal. This had led to a variety of indirect enzymic methods of analysis, where the activity of the enzyme results in the conversions between NAD+ and NADH. These are discussed in Sec. 8. 

Synthesis of sucrose 6 -phosphate by an enzymic method using uridine 5 -(a-D-glucopyranosyl diphosphate) plus D-glucose 6-phosphate has been reported.126-131 The first, unambiguous, chemical synthesis of sucrose 6 -phosphate was achieved by Buchanan and coworkers.18 The reaction of 2,3,4,6,l, 3, 4 -hepta-0-acetylsucrose, prepared by five steps of synthesis, with cyanoethyl phosphate in pyridine gave a crude product from which sucrose 6 -phosphate was isolated as the barium salt. 

Enzymic methods in preparative carbohydrate chemistry, 49, 175-237 Enzyme specificity, how Emil Fischer was led to the lock and key concept for, 50, 1-20... 

Fonong T. 1987. Enzyme method for the spectrophotometric determination of micro-amounts of cyanide. Analyst 112 1033-1035. 

Similarly with regard to the fluoroacetate story, other factors in addition to the jamming of the Krebs cycle may be looked for. One profitable line may well be the examination of the mobility, by chemical and enzyme methods, of the firmly... 

Enzymic methods for the quantitation of monosaccharides are employed when a higher degree of specificity is required than can be achieved by the majority of the chemical methods. They often enable the quantitation of one stereoisomer in the presence of others and can often differentiate between the a and 0 anomeric forms. 

Two other commonly occurring hexoses which are usually found as components of polysaccharides or combined with other molecules in complex structures are galactose and fructose and, in a similar manner to other monosaccharides, enzymic methods are available for their measurement. An enzymic method for the measurement of fructose using hexokinase was described earlier, together with the method for mannose and glucose (Figure 9.22). 

The chemical methods for the quantitation of cholesterol measure total cholesterol, i.e. free and esterified, and so a digitonin precipitate must be prepared if free cholesterol is to be measured. Enzymic methods do not measure the esters and a hydrolysis stage, either chemical or enzymic (using cholesterol ester hydrolase, EC 3.1.1.13), is necessary for the measurement of total cholesterol. 

Procedure 12.2 Enzymic method for the quantitation of free cholesterol... 

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