Enzyme standards, electrophoresis

Enzyme Standards. Enzyme electrophoresis has proliferated with Increased genetic profiling for medical and forensic use. Standards for enzyme analyses In the form of defined kits containing banks of enzymes of different Isoenzyme patterns are not commercially available. For the most part, forensic laboratories use Individuals from within their own laboratories who have known phenotypes for the enzymes of Interest. Such donors become de facto standards. The Interlaboratory exchange of samples and rigorous continued training In enzyme phenotype Identification will Improve this standards base. 

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. 

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

In vitro. The activity or absolute level of enzymes such as cytochrome P-450 and glucuronosyl transferase can be measured in cells, tissue fractions, or subcellular fractions (e.g., microsomes) and compared with those from control animals. The activity is measured by using a particular substrate for each of the isoforms of the enzyme (e.g., cytochrome P-450 or UDPGT) of interest. The total level of cytochrome P-450 could be determined by spectrophotometry using standard methods (e.g., carbon monoxide binding and difference spectra). Alternatively, the level of protein can be determined by gel electrophoresis and Western blotting, and this would allow the separation of different isoforms. 

Creatine kinase was purified from rabbit muscle by the method of Kuby et al, (4). Rabbit muscle pyruvate kinase was purchased from Boehringer. Porcine muscle adenylate kinase was purchased from Sigma, and was further purified by gel filtration on Sephadex G-50. The enzymes were homogeneous as judged by their specific activities and by their migration as single components in sodium dodecyl sulfate gel electrophoresis. Proton NMR spectra at 250 MHz of 0.5-2.0 mM enzyme sites in 0 solution were obtained with a Bruker WM 250 MHz pulse FT spectrometer at 25°. At least 256 transients were accumulated over 8192 data points using 16 bit A/D conversion. Relaxation rates and histidine pK values were determined by standard NMR methods (5, 6),... 

Aussenac et al. (1998) used capillary zone electrophoresis with UV detection at 260 nm to analyze isoflavones in soybean seeds of various varieties grown in various locations. Methanol was used for extraction. Total extraction was not affected by temperature but was affected by the composition of the solvent. Electrophoresis was conducted at pH 10.5, at which the isoflavones were weak acids and were ionized. Boric acid was added to form a negatively charged borate-isoflavone complex. A fast capillary electrophoresis method was also developed by Vanttinen and Moravcova (1999) to determine daidzein and genistein after enzyme hydrolysis in soy products. Photodiode array was used to detect the isoflavones at 254 and 268 nm, respectively. Minimum detection was 0.4 mg/L. p-Nitrophenol was used as an internal standard. 

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