Electrophoresis enzyme purification

Affinity chromatography, based on biological recognition, has become a major means for the purification of biologically active molecules 2). The technique provides a simple and effective way of purification. Specific adsorption of the enzyme to its competitive inhibitor attached to a polymer matrix is the basis for an efficient enzyme purification. Affinity electrophoresis is also based on biological recognition3 . 

A NAD+-dependent (S)-l,3-BDO dehydrogenase (designated as CpSADH), which appears to participate in the production of (R)-1,3-BDO from the racemate, was purified from C. parapsiiosis IFO 1396 [13]. The results of the enzyme purification are summarized in Tab. 3. The specific activity of the final preparation was about 5400-fold higher than that of the crude extract. The molecular weight of the enzyme was estimated to be approximately 40,000 by SDS-polyacrylamide gel electrophoresis and approximately 140,000 by gel filtration. The enzyme showed the maximal activity at 50°C and pH 9 for the oxidation of (S)-l,3-BDO. 

Many of the methods described in Chapter 4 are used to purify enzymes in their native state. Why would the use of SDS-polyacrylamide gel electrophoresis be unlikely to lead to the successful purification of an active enzyme What experiments would you conduct to determine whether salting out with ammonium sulfate would be useful in enzyme purification ... 

Electrophoresis also provides a useful technique in biosynthesis for protein and enzyme purification and production. 

The steps of enzyme purification were carried out first by polyethylene glycol 6000 (PEG) concentrations of the 100,000 g supernatants according to Chayet at at, 0977). The PEG precipitates (30- 0%) were separated on DEAE-Sephacel. This separation was followed by a chromatography on an affinity column using Sepharose-Aminophenethyl pyrophosphate (Dogbo and Camara, 1987). Electrophoresis on poly-... 

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

Proteolytic enzyme from the latex of Carica papaya with an approximate molecular weight of 27000. It is differentiated from papain in electrophoresis behavior, in solubility and in substrate specifity. Isolation by acidify of papaya-latex with HCl, salting out with NaCl and following chromatographic purification. The formulation contains L-cysteine as reducing agent. 

Figure 5 SDS Polyacrylamide Gel Electrophoresis of pectinase from different steps of purification A (1,6) standard protein (2) crude enzyme (3)...

Miller and Macmillan [4] carried out purification of pectinesterase from Fusarium oxysporum f. sp. vasinfectum culture fluid (fivefold degree of purification). According to the obtained data the purified enzyme possessed very low polygalacturonatlyase one. Disk electrophoresis at pH 4.3 revealed two protein components. The authors did not study distribution of pectinesterase activity in these components. Molecular weight of fungal pectinesterase determined using gel — filtration on Sefadex G — 75 was found to be 35,000. 

In the absence of K the enzyme exhibits a basal Mg -ATPase activity that can be reduced, but not completely removed, upon further purification of the enzyme by free-flow or zonal electrophoresis [66,89]. Wallmark et al. [104] demonstrated that the rate of spontaneous breakdown of phosphoenzyme corresponded very well to the Mg -ATPase activity at low ATP concentrations, implying that this activity was not due to a contaminating Mg -ATPase with a reaction path independent of the phosphoenzyme. This conclusion was confirmed by Reenstra et al. [129] in a study on the nonhyperbolic ATP dependence of ATPase activity and phosphoenzyme... 

For CBCA synthase hardly any information has been pubhshed. The enzyme was characterized after it was purified from C. sativa extracts and until this moment no sequence has been deposited. After purification of the protein it was found to be a homodimeric enzyme, meaning that enzyme is formed by two identical domains. This was observed after the purification, when the enzyme had a molecular weight of 136 kDa, and after denatured electrophoresis, when it had a molecular weight of 71 kDa. Furthermore, the CBCA synthase has shown to bear higher affinity for CBGA (1717 M S ) than THCA synthase and CBDA synthase (respectively 1382M s and 1492 M s ), which is probably due to its homodimeric nature [40]. 

As metabolic pathways became clearer, the detailed study of the enzymes involved was facilitated by the introduction of new procedures for isolation, purification, and characterization of proteins. Developments in chromatography in the early 1940s and the introduction of gel electrophoresis allowed more efficient methods to be used to separate proteins and to analyze their primary structure, so that Sanger was able, by 1953, to report the primary structure of insulin (Chapter 10). 

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... 

Table 10.3 contains a listing of some important proteins. Protein purification must be done under conditions in which conformational and configurational changes are minimal. Such purification is most often carried out using varieties of chromatography including affinity chromatography and electrophoresis. Some of the common features of enzymes are ... 

Only a few bacterial and viral sialidases have been purified to high purity or even to protein homogeneity."0 Complete purification of sia-lidase on a preparative scale from the culture filtrate ofC. perfringens was achieved111 by using poly(acrylamide) gel-electrophoresis as the final purification step (see Section VI,1). It is necessary that such purified sialidases be available, as the presence of proteases or other gly-cosidases in the enzyme preparations would lead to severe errors, not only in studies of substrate specificity, but also in cell biological and medical studies (see Sections VI and VII). 

Takazawa, K. Passareiro, H. Dumont, J.E. Erneux, C. Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Biochem. J., 261, 483-488 (1989)... 

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