Automated enzyme analysis

Methods of Assay. — The Technicon basic autoanalyser sampler system has been modified for simultaneous sampling of glycoside hydrolases and substrate-buffer solutions. This inexpensive modification allows performance of automated enzyme analysis and enzyme kinetic studies with minimal consumption of substrate and/or enzyme. 

The problems of enzyme analysis provide a good example of the development of an automated system. There are three stages in the auto-... 

Rodbard D, McClean SW. Automated computer analysis for enzyme-multiplied immunological techniques. Cfin Chem 1977 23 112-5. 

Eisenthal R, Peterson ME, Daniel RM et td. (2006) The thermed behaviour of enzyme activity impheations for biotechnology. TIBTECH 24(7) 289-292 Endo J, Tabata M, Okada S, Murachi T (1979) Use of immobilized enzymes in automated clinical analysis determination of uric acid and glucose using immobilized enzymes in column form. Clin Chim Acta 95(2) 411-417. 

In order to automate the analysis, these methods frequently combine immobilized enzymes with flow or sequential injection techniques. These methods may include a separation step such as solid-phase extraction, gas diffusion, or pervaporation. The latter is a nonchromatographic separation technique, which selectively separates a liquid mixture by partial vaporization through a nonporous polymeric membrane. Separation is not based on relative volatilities as in distillation, but rather on the relative rates of permeation through the membrane. 

The connection of biochemical reaction with electrochemical detection sometimes causes difficulties, for example if the conditions of the biochemical reaction and detector function are different [317, 318]. In such cases it is advantageous to carry out the biochemical reaction separately and to determine the reaction product after changed conditions [319]. Such an enzyme reactor electrode enables a large choice of detection methods, kinds and forms of applied biologically active materials, number of analyses provided per hour and possibilities of automation. Enzyme reactor electrodes are therefore used not only in the above characterized difficult cases, but are often preferred in automated methods of analysis. 

Methods of Assay.—Included in a handbook on methods of enzymic analysis are methods of assay, automated assays, uses of immobilized enzymes, and sources of enzymes and reagents. The latest volume in the series Principles of Enzymatic Analysis introduces a new nomenclature for quantities and units of activity, and discusses in detail prerequisites for the development of new assays. ... 

To test the viability of the method for such applications, 12 hydrolytic enzymes were investigated with two automated multiple enzyme analysis systems [46]. Hydrolases in continuous-flow systems were successfully appHed to determine glycogen without interference from free glucose [47] and for the determination of total serum cholesterol [48]. Microreactor technology combined with a hydrolase-based enzymatic method was applied for high-throughput optimization of the HCN addition to aldehydes by enantioselective Lewis acid/Lewis base catalysis [49]. 

Introduction 27.6 Enzyme Digestion of 27.12 Automated Particle Analysis... 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Moody, G.C. et al. 1999. Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes. Xenobiotica. 29 53. 

Moody, G.C., Griffin, S.J., Mather, A.N., McGinnity, D.F. and Riley, R.J. (1999) Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes assessment of human CYP inhibition potential. Xenobiotica, 29 (1), 53-75. 

Atkinson, A., Kenny, J.R. and Grime, K. (2005) Automated assessment of time-dependent inhibition of human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometry analysis. Drug Metabolism and Disposition, 33 (11), 1637—1647. 

Biomedical analytical chemistry happens to be one of the latest disciplines which essentially embraces the principles and techniques of both analytical chemistry and biochemistry. It has often been known as clinical chemistry . This particular aspect of analytical chemistry has gained significant cognizance in the recent past by virtue of certain important techniques being included very much within its scope of analysis, namely colorimetric assays, enzymic assays, radioimmunoassays and automated methods of clinical analysis. 

The use of spectrophotometry to monitor enzyme-catalysed reactions (Table 8.6) is a very convenient and popular method owing to the simplicity of the technique and the precision that is possible. The technique lends itself readily not only to temperature control using water-jacketed or electrically heated cell holders but also to the measurement of initial velocities by continuous monitoring and recording techniques or by automated analysis systems. 

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