Enzyme activation using

Other analyzers such as the Gilford Automated Enzyme Analyzer and the LKB-8600 Reaction Rate Analyzer analyze discrete samples one at a time. These instruments provide kinetic analyses, digital data reduction at the time each sample is analyzed, and excellent electronic and optical characteristics. Recently, Atwood has developed kinetic enzyme analyzers which require only 9 seconds for measuring an enzyme activity, using highly stable and sensitive electronic circuits (12). This short read out time allows a large number of samples to be processed by one instrument in an automated mode. 

Many automated instruments measure enzyme activity using fixed time colorimetric methods. Some, however, can be classed as reaction rate analysers, e.g. the centrifugal analysers, and these instruments determine the reaction rate from either the initial slope of the reaction curve or from repeat measurements at fixed intervals. In both methods the slope of the line is taken to represent the activity of the enzyme. 

The presence of a lag period in many coupled assays and difficulties in determining the linear portion of a curve present the main problems in the calculation of enzyme activity using reaction rate analysers. In the simplest instruments the slope of the curve in the first few seconds of the reaction is extrapolated into a straight line or, if the reaction is known to show a lag period, the rate of reaction after a defined period of time can be measured. The more sophisticated instruments use microcomputers to determine the linear portion of the curve and calculate the enzyme activity directly from the slope. The second derivative of the reaction progress curve (rate of change of the slope) can be monitored by the computer and when a value of zero is held for a period of time (10—15 seconds) this indicates a linear section of the graph. From the value for the slope, the enzyme activity can be calculated. 

Flour, soya, enzyme-active, use in making white bread, 192-198 Food(s)... 

Fig. 31.9. Relationship between Penicillin G Acylase loading level and on-bead enzyme activity using Amberzyme Oxirane resin loadings at higher challenge levels. (Courtesy of M. Elizabeth Miller and Rohm and Haas.)...

Calculation One Glucose Oxidase Titrimetric unit of activity (GOTu) is the quantity of enzyme that will oxidize 3 mg of glucose to gluconic acid under the conditions of the assay. Determine the enzyme activity using the following equation ... 

Assay each of the fractions preserved in steps 10-5 to 10-38 for protein concentration and enzyme activity. Use the Lowry method described in Chapter 2 for protein determinations. Use the enzyme assay procedures described in steps 10-52 to 10-57 to determine enzyme activity. 

Moore BD, Stevenson L, Watt A, Flitsch S, Turner NJ, Cassidy C, Graham D (2004) Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering. Nat Biotechnol 22 1133-1138... 

In most amperometric cytochrome b2 electrodes the reaction is followed by anodic oxidation of ferrocyanide at a potential of +0.25 V or above. The first of such sensors was assembled by Williams et al. (1970), who immobilized the enzyme (from baker s yeast) physically at the tip of a platinum electrode within a nylon net of 0.15 mm thickness. The large layer thickness resulted in a response time of 3-10 min. Owing to the low specific enzyme activity used, the sensor was kinetically controlled. Therefore the linear measuring range extended only up to 0.1 Km-A similar sensor has been applied by Durliat et al. (1979) to continuous lactate analysis. The enzyme was contained in a reaction chamber of 1 pi volume in front of the electrode. This principle has also been employed in the first commercial lactate analyzer using an enzyme electrode (Roche LA 640, see Section 5.2.3.3X With a sensor stability of 30 days and a C V below 5%, 20-30 samples/h can be processed with this device. 

The previous discussion has illustrated the role of monooxygenase systems in fish for the metabolism of xenobiotics. Measurements of this activity have therefore been used as a measure of the extent to which fish have been exposed to xenobiotics at the same time, of course, increased levels enable the fish to metabolize xenobiotics effectively (Kleinow et al. 1987). Although specific assays of cytochrome P-450 activity may be made by immunoblot methods (Monosson and Stegeman 1991), it may be expedient to measure specific enzyme activity using defined substrates. Two assays have been widely used (1) aryl hydrocarbon hydroxylase activity that may be assayed using benzo[a]pyrene as substrate, although this substrate has been replaced recently by the less hazardous 2,5-diphenyloxazole, and (2) activity for O-deethylation of 7-ethoxyresorufin (EROD) has been extensively used and is a simple and convenient assay. 

Stop the enzyme reaction by adding 25 pL stop solution and measure the enzyme activity using a microtiter plate reader at A450nm. 

Gels are stained for proteins with oomassie brilliant blue R 250 by the usual procedure. Alternatively, gels can be stained for enzyme activity using specific staining methods. 

Evans, G. O. 1989. Species relationships for plasma angiotensin converting enzyme activity using a furanacryloyl tripeptide substrate. Experimental Animals 38 897-898. 

Mermelstein, L.D., Papoutsakis, E.T., Petersen, D.J., and Bennett, G.N. (1993) Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon. Biotechnol. Bioeng., 42, 1053—1060. Qureshi, N., Li, X.-L., Hughes, S, Saha, B.C. et al. (2006) Butanol production from com fiber xylan using Clostridium acetobutylicum. Biotechnol. Progr, 22, 673 -680. 

Probing DNA Repair Enzyme Activity Using Independently Generated 133 2 -Deoxyuridin-1 -yl Radical (3)... 

In this entry, the authors present an overview of the LC-MS of naturally occurring steroids in biological samples, which was published from January 2001 to June 2006. Steroids are divided into six categories, based on their stmctures and functions, and no attention has been paid to steroid hormones used as growth promoters of animals and used in sports doping. This entry also does not take up references on the in vitro assays for the measurement of enzyme activity using a steroid as the substrate. 

The crude fish enzyme extracts were prepared as per the method of Baranowski et al. (1984), and stored in ice for use in the pressure treatments and subsequent enzyme assays. The spectrophotometric methods of Hummel (1959) and Erlanger et al (1961) were used to assay for chymotrypsin-like and trypsin-like enzyme activities using BTEE and BAPNA as substrates, respectively. Cathepsin C activity was assayed using gly-phe-NA as substrate (Lee et al, 1971), and collagenase activity in the fish extracts were assayed as per the method of Wunsch and Heidrich (1963). Protein content of the crude enzyme extracts from fish was determined by the method of Hartree (1972). 

The basis for the detection of enzyme activities using 4-methyl-umbelliferyl glycosides is the release of the glycon, 4-methylumbelli-ferone, which shows an intense fluorescence in UV light 350 nm) [6]. 

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