Enzyme separation, electrophoretic

Techniques have also been developed for the specific visualization of particular classes of enzymes following electrophoretic separation in a gel. These techniques are often referred to as activity staining, as the intrinsic activity of the enzyme is used, either to produce a colored product or to produce a clear zone on a colored background within the gel. A method for visualizing proteinases based on the work of Gar-cfa-Carreno and Haard (1993) and Garcia-Car-reno et al. (1993) is presented (see Basic Protocol 3). 

Isoenzymes Isoenzymes are different forms of an enzyme which catalyze the same reaction, but which exhibit different physical or kinetic properties. The isoenzymes of lactate dehydrogenase (LDH) can be separated electrophoretically and can be used clinically to diagnose a myocardial infarction. 

The enzyme LDH is a tetramer of two different subunits, called H and M. The predominant subunit in heart cells is H and the predominant subunit in skeletal muscle or liver is M. The isoenzymes contain different proportions of the two subunits, that is, H4, H3M, H2M2, HM3, and M4. The isoenzymes can be separated electrophoretically. 

Creatine kinase (CK or CPK) is one of these enzymes. The protein is composed of two subunits, which may be either of the muscle (M) or the brain (B) type. The MB form, containing one M and one B subunit, is found primarily in cardiac muscle. It can be separated electrophoretically from other CK isozymes and the amount in the blood used to determine if a myocardial infarction has occurred. On admission to the hospital, Ann Jeina s total CK was 182 units/L (reference range 5 38-174 U/L). Her MB fraction was 6.8% (reference range 5% or less of the total CK). Although these values are only slightly elevated, they are typical of the phase immediately following a myocardial infarction. Additional information was provided by myoglobin and troponin T (Tn-T) measurements. 

Loureiro, 2000 Deak, 2002 Capece et al., 2003). Each requires initial digestion of the harvested DNA using restriction endonucleases, enzymes that cleave DNA at specific nucleotide sequences unique for that enzyme. The DNA digest is the amplified at specific or randomly selected regions by polymerase chain reaction (PCR). Fragments are subsequently separated electrophoretically and their patterns compared against those of other isolates or databases. 

There have been two approaches to the routine determination of isoenzyme activity in serum. The first is the separation of the enzymes by electrophoretic means on a variety of media including starch, agar, polyacrylamide and cellulose acetate. 

This is most usually achieved with buffers. Enzyme reactions, electrophoretic separations and also spectrometric and electrochemical determinations may aU require pH control. 

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. 

Glucose-6-phosphate dehydrogenase is a dimer with a molecular mass of about 135 000. Up to eight electrophoretically separable isoenzymes for this enzyme are known. A specific feature of the above reaction is the formation of NADP Hr The reaction equilibrium is strongly shifted to the right, since the lactone formed is liable to hydrolysis, which is spontaneous or lactonase-assisted. 

An interesting twist to the story is provided by studies on N. brasiliensis, which secretes three distinct isoforms of AChE, designated A, B and C (Ogilvie et al, 1973). These enzymes can be easily separated by nondenaturing electrophoresis due to their distinct pis, and this is illustrated in Fig. 11.1, which also shows the distinct electrophoretic properties of the amphiphilic enzyme (arrowed) found only in somatic extracts and therefore presumably associated with neuromuscular function. The overall amount of AChE produced by this parasite increases dramatically following establishment in the jejunum, and a switch in isoform expression occurs,... 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

Acid dye method for the analysis of thiamin, 18A, 73 electrophoretic separation and fluorometric determination of thiamin and its phosphate esters, 18A, 91 catalytic polarography in the study of the reactions of thiamin and thiamin derivatives, 18A, 93 preparation of thiamin derivatives and analogs, 18A, 141 preparation of the mono- and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine for thiamin biosynthesis, 18A, 162 formation of the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine by enzymes from brewers yeast in thiamin biosynthesis, 18A, 203 resolution, reconstitution, and other methods for the study of binding of thiamin pyrophos-... 

Five- to six-month-old tobacco plants (Nicotiana tabacum var. Samsun) grown in a glasshouse at 20°C were used for this study. Commercial synthetic substrates employed both for histochemical and biochemical assays were guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), 3-3 di-aminobenzidine (DAB), tetramethylbenzidine (TMB) and syringaldazine. Isopropylamine and monosodium salts of ferulic acid were also used as substrates as well as their / -fluorinated analogues substituted with a fluorine atom on the / -carbon (Fig. 1). Histochemical observations were done on hand-made transverse sections of fresh tobacco stems. Biochemical assays were performed separately on bark (inner cortical parenchyma, phloem and fibres) and xylem fractions. Technical data of incubation, enzyme extraction, spectrophotometric and electrophoretic assays were given elsewhere (5-7). Synthesis of fluorinated compounds was performed as previously described (4). 

The culture filtrates of T. reesei contained a large number of both cellulolytic and hemicellulolytic enzymes, which could be partially separated by chromatofocussing (Fig. 1). Of the cellulolytic enzymes, several endoglu-canases and two cellobiohydrolases have already been isolated and characterized (30). Some of the endoglucanases isolated are nonspecific and have xylanase activity (31). The two major xylanase (Xyl) peaks in Figure 1 corresponded to pi-values of above 7.5 and 5.5. When the former enzyme was further purified (24), its isoelectric point was found to be about pi 9 (Table I). Previously, the presence of at least three electrophoretically different xylanases in T. reesei culture filtrates was reported, with the most acidic one shown to have broad substrate specificity (10). This is in agreement with the reported occurrence of three xylanases (pi > 7, pi 5.1, pi... 

Shovers, J., Fossum, G. and Need, A. 1972. Procedure for electrophoretic separation and visualization of milk-clotting enzymes in milk coagulants. J. Dairy Sci. 55, 1532-1534. 

Lactate dehydrogenase exists in the cytoplasm of humans and most animals as five forms which are easily separable by electrophoresis and are evenly spaced on electropherograms.8 This enzyme is a tetra-mer made of two kinds of subunits. Isoenzyme 1, which has the highest electrophoretic mobility, consists of four identical type B subunits. The slowest moving tetramer (isoenzyme 5) consists of four type A subunits, while the other three forms, AB3, A2B2, and A3B, contain... 

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