Enzymes analysis with

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. 

Kinetics is the branch of science concerned with the rates of chemical reactions. The study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they accomplish their remarkable feats. In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors. Coupled with studies on the structure and chemistry of the enzyme, analysis of the enzymatic rate under different reaction conditions yields insights regarding the enzyme s mechanism of catalytic action. Such information is essential to an overall understanding of metabolism. 

Gray, R. "Sequence Analysis with Dansyl Chloride", In "Methods In Enzymology", p. 333, Vol. XXV, "Enzyme Structure, Part B", C. H. W. Hlrs and S. N. Tlmasheff, Editors, Academic Press, New York, 1972. 

J.W. King and K.S. Nam, Coupling enzyme immunoassay with supercritical fluid extraction, in Immunoassays for Residue Analysis, ed. R.C. Beier and L.H. Stanker, American Chemical Society, Washington, DC, Chapter 34, pp. 422-438 (1996). 

Reverse genetics has been applied to diseases such as Duchenne muscular dystrophy and cystic fibrosis, in which the responsible enzymes are unknown and the disease results from a significant deletion. By combining RFLP analysis with cytogenetics, it has been possible to increasingly narrow the location of the defective genes to small regions on the affected chromosomes. 

Electrophoretic and isotachoelectrophoretic techniques are gaining in popularity in soil analysis with applications to polyaromatic hydrocarbons, polychlorobiphenyls, tetrahydrothiophene and triazine herbicides, Paraquat and Diquat and growth regulators. Other lesser-used techniques include spectrophotometric methods (five determinants), spectrofluorimetric methods (two determinants), luminescence methods (one determinant), titration methods (one determinant), thin-layer chromatography (five applications), NHR spectroscopy (two applications) and enzymic immunoassays (one determinant). 

We employed various substrates to check for MFO in two bivalve species, a salt water mussel (Mytilus edulis) and a fresh water clam (Anodonta sp). Cytochrome P-450 was also studied. Organisms were exposed to 100 PPM Venezuelan crude in a stagnant system for up to one month. Enzyme assays were carried out with digestive gland 9000 g homogenates (17) and cytochrome P-450 analysis, with microsomes (21). The hydrocarbon substrates investigated included 1I+C-labelled benzo(a)pyrene, fluorene, anthracene, and naphthalene. The method used for separation of BP metabolites by thin layer radiochromatography has been described (7). The metabolite detection method for the other aromatic hydrocarbons was essentially the same except methylene chloride was used as metabolite extractant as well as TLC developer. Besides the hydrocarbon substrates, we also checked for other MFO reactions, N-dealkylase with C-imipramine (22) and 0-dealkylase with ethoxycoumarin (15). 

The principle behind the test method(s) is that antibodies are made of proteins that recognize and bind with foreign substances (antigens) that invade host animals. Synthetic antibodies have been developed to complex with petroleum constituents. The antibodies are immobilized on the walls of a special ceU or filter membrane. Water samples are added directly to the cell, while soils must be extracted before analysis. A known amount of labeled analyte (typically, an enzyme with an affinity for the antibody) is added after the sample. The sample analytes compete with the enzyme-labeled analytes for sites on the antibodies. After equilibrium is established, the cell is washed to remove any um-eacted sample or labeled enzyme. Color development reagents that react with the labeled enzyme are added. A solution that stops color development is added at a specified time, and the optical density (color intensity) is measured. Because the coloring agent reacts with the labeled enzyme, samples with high optical density contain low concentrations of analytes. Concentration is inversely proportional to optical density. 

In some cases, an inhibitor can bind to more than one site on an enzyme protein, with inhibition resulting from binding at multiple sites. Binding affinities at the two (or more) sites may be different, and mechanisms of inhibition may be dilferent for example, high-affinity inhibition might occur through an allosteric site and lower affinity inhibition through the active site. Analysis of such systems is complex and may require a combination of several of the approaches outlined later. 

Competitive, 249, 123, 146, 190 [partial, 249, 124 progress curve equations for, 249, 176, 180 for three-substrate systems, 249, 133, 136] competitive-uncompetitive, 249, 138 concave-up hyperbolic, 249, 143 dead-end, 249, 124 [for bireactant kinetic mechanism determination, 249, 130-133 definition of kinetic constants, 249, 220-221 effects on enzyme progress curves, nonlinear regression analysis, 249, 71-72 inhibition constant evaluation, 249, 134-135 kinetic analysis with, 249, 123-143 one-substrate systems, 249, 124-126 unireactant systems, theory,... 

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