Spectrophotometric assays

PAL activity is assayed spectrophotometricaUy based on the methods of Koukol and Conn (1961) and Kataoka et al. (1983) by measurement of the rate of formation of t-cinnamic acid as the increase in absorbance at 268 nm. The reaction mixture consists of 2 ml of the enzyme preparation and 1ml of 25 pm l-phenylalanine. In the reference mixture, the L-phenylalanine solution is replaced by distilled water. The mixture is incubated at 37 °C for 3h with shaking. The reaction is stopped by the addition of 0.1 ml of 6 N HCl. After 5 ml of peroxide-free ethyl ether is added, the mixture is shaken again vigorously for extraction of the acidified fraction and then centrifuged at 3000 g for 5 min. The ether phase is obtained with a pipette, and the ether is evaporated to dryness under reduced pressure. The residue is dissolved in 4 ml of 0.05 N NaOH and the absorbance of the solution at 268 nm in a 1-cm quartz cell is measured. The enzyme activity is expressed as micromoles of t-cinnamic acid formed per g FW or per mg protein in fresh tissue/h. The reaction product is scanned with a spectrophotometer for comparison of its optical characteristics with those of authentic t-cinnamic acid. The absorption spectrum of the reaction product was identical with that of authentic f-cinnamic acid (Fig. 3). 

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The British Pharmacopoeia specifies a biological assay for the sodium salt of rifamycia SV [14897-39-3]. It also specifies a spectrophotometric assay for rifampicia (201). The United States Pharmacopeia requires an hplc assay for rifampin (202). 

An example of a direct spectrophotometrical assay is the use of synthetic peptide -nitroanilide substrates to determine protease activity. The /)-nitroani1ine group Hberated from the substrates by the protease can be determined spectrophotometricaHy at 410 nm. An example of an indirect (coupled) spectrophotometric assay is the determination of a-amylase using -nitrophenyLmaltoheptaoside. Initially, the substrate is cleaved by the a-amylase and subsequentiy one of the reaction products, -nitrophenyLmaltotrioside, is cleaved by a-glucosidase, hberating -nitrophenyl, a chromophore... 

Application of IP and NCS in conjunction with specification tolerance limits enables to substantiate acceptance criteria for linear regression metrological characteristics (residual standard deviation, correlation coefficient, y-intercept), accuracy and repeatability. Acceptance criteria for impurity influence (in spectrophotometric assay), solution stability and intermediate precision are substantiated as well. 

Developed standai d validation procedure is demonstrated for validation of a spectrophotometric assay of ambroxol hydrochloride tablets. Without any considerable revisions, this approach may be applied to chromatographic methods. Recommendations for validation criteria were included in the State Phai macopoeia of Ukraine. 

The physicochemical properties of the reactants in an eiKyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the abihty of a substrate or product to absorb hght. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb hght at a wavelength of 340 run, whereas their oxidized forms NAD(P) do not (Figure 7—9). When NAD(P)+ is reduced, the absorbance at 340 run therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 run will be observed. In each case, the rate of change in optical density at 340 nm will be proportionate to the quantity of enzyme present. 

Comparative studies of the widely employed spectrophotometric readings at the Soret and Q bands (405 and 630 nm, respectively) and the elemental analysis of copper and nitrogen showed that the spectrophotometric assay based only on the Soret band can overestimate the purity of a preparation. Erroneous data were attributed to an increase in absorptivity at the Soret band when other colored compounds like metal-free analogs and carotenoids are present. Indeed, copper-free chlorin e6 exhibits a specific absorbance 3.6 times greater than that of its coppered counterpart. Therefore, measurements at the Q band (630 mn) and the establishment of the S Q ratio are preferred. 

Sodium copper chlorophyllin, approved by the FDA as a color additive in citrus-based dry beverage mixes, should have a ratio of absorbance (SoretQ band) not less than 3.4 and not more than 3.9. In Europe, purity criteria of the food additives E141[i] and E141[ii], which are copper complexes of chlorophyll and chlorophyllin, respectively, are set out in the EC color specifications that include identification and spectrophotometric assay tests. ... 

A rapid second-derivative spectrophotometric assay procedure was described for the simultaneous determination of niclosamide and thiabendazole in tablets [47]. The derivative absorbances were measured at 354 nm for niclosamide and at 242.5 nm for thiabendazole. 

Prasad et al. [17] recommended a spectrophotometric assay method for the determination of primaquine in plasma. 

Crook EM, Mathias AP, Rabin BR. Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2, 3 -phosphate. Biochem. J. 1960 74 234-238. 

Green, N.M. (1965) A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J. 94, 23c-24c. 

Spectrophotometric Assays for the Determination of Specific Phenolic Groups... 

Valkonen M and Kuusi T. 1997. Spectrophotometric assay for total peroxyl radical-trapping antioxidant potential in human serum. J Lipid Res 38(4) 823-833. 

Oberzill has described a spectrophotometric assay for gramicidin156. Thombs and coworkers have used the... 

In this method the reaction of diphenylpicrylhydra-sine with methimazole (thiamazole) was sufficiently rapid for its use in a spectrophotometric assay. 

More recently Agrawal, Harmalker and Vijayawargiya9 have described the formation of a copper-neomycin complex. With a stoichiometry of 1 1, the blue coloured complex has been made the basis of a spectrophotometric assay method for neomycin(See Section 6.26). 

Spectrophotometric assays of neomycin may be conveniently divided into two groups -... 

The manometric method using the Warburg apparatus is highly accurate, but complicated, and demands more time than the simple spectrophotometrical assay, its accuracy being equal. 

Gentest (now BD Biosciences) was the first to develop spectrophotometric assays to study CYP inhibition [98]. These assays are based on the turnover of mildly fluorescent substrate probes to moderately fluorescent metabolites, where metabolite formation is monitored by an increase in fluorescence using a plate reader [99,100]. Problems with these methods include background interference due to low signal-to-noise ratio, chemotype-specific interference and fluorescence quenching. Aurora Biosciences (now Vertex) has designed probes that exhibit larger fluorescence... 

Prior to the genomics era, most metabolite measurements were performed in targeted approaches such as spectrophotometric assays or HPLC with UV detection. Today, these methods are still the best choice for the analysis of small numbers of metabolites. In spectrophotometric assays, metabolites are first... 

This is a spectrophotometric assay based on the reaction of diphenylamine with the deoxyribose moiety of DNA to produce a complex that absorbs at 600 nm. The reaction is specific for deoxyribose and RNA does not interfere. It can be used on relatively crude extracts where direct spectrophotometric determinations of DNA concentration are not possible. 

TK activity was determined by a spectrophotometric assay at 340 nm. In 0.5 mL of tris buffer (50 mM) pH 7.6, were added 50 pL of L-erythrulose from a 1 m stock solution in water (0.05 mmol), 25 pL of o-ribose-5-phosphate from a 160 him stock solution in water (4.0 pmol), 5 pC of ThDP from a 21 mM stock solution in water (0.1 pmol), 10 pL of MgCl2 from a 50 mM stock solution in water (0.5 /rmol), 10 pL of NADH from a 14 him solution in water (0.14 /imol), alcohol dehydrogenase (12 units). 

A rapid dilution procedure is routinely used in the author s laboratory to assess reversibility, and it is particularly enlightening if enzyme activity is then determined in a continuous spectrophotometric assay. A microplate assay is set up, in triplicate, as outlined in Table 4-3, later. It is assumed, for the purposes of this example, that the for substrate is 10 tM and that the IC50 for the inhibitor in the presence of 10 tM substrate is 200 tM. The concentration of enzyme used in control wells should be at least tenfold greater than the minimum concentration necessary to catalyze a quantifiable increase in product concentration over the duration of the incubation of substrate with enzyme. 

The mean sizes obtained were 500 150, 350 77, 192 25, and 100 16, for 0.6, 0.4, 0.2, and 0.1pm liposomes, respectively. Drug concentration was determined by spectrophotometric assay of chromophoric complex between the BP and copper (II) ions (63) or by high performance liquid chromatography (HPLC) (64). Lipid concentration was determined by Bartlett method (65). Stability of the liposomes was determined by examining drug leakage. Then 400 pL of liposomal formulations were centrifuged... 

Fatkins, D.G. and Zheng, W. (2008) A spectrophotometric assay for histone deacetylase 8. Analytical Biochemistry, 372, 82-88. 

After cleaning with a phosphate-free detergent, labware should be rinsed extensively with deionized water and finally rinsed at least twice with doubly distilled water before drying. Avoid using paper towels, because lint and microscopic paper fibers often contain metal ions as well as acids/bases used in commercial treatment of paper products. Paper fibers also scatter light and interfere with spectrophotometric assays. 

Enzyme Assays. Procedures for the HPLC assay of prephenate aminotransferase (32), the spectrophotometric assay of shikimate dehydrogenase... 

Reflectance measurements provided an excellent means for building an ammonium ion sensor involving immobilization of a colorimetric acid-base indicator in the flow-cell depicted schematically in Fig. 3.38.C. The cell was furnished with a microporous PTFE membrane supported on the inner surface of the light window. The detection limit achieved was found to depend on the constant of the immobilized acid-base indicator used it was lO M for /7-Xylenol Blue (pAT, = 2.0). The response time was related to the ammonium ion concentration and ranged from 1 to 60 min. The sensor remained stable for over 6 months and was used to determine the analyte in real samples consisting of purified waste water, which was taken from a tank where the water was collected for release into the mimicipal waste water treatment plant. Since no significant interference fi-om acid compounds such as carbon dioxide or acetic acid was encountered, the sensor proved to be applicable to real samples after pH adjustment. The ammonium concentrations provided by the sensor were consistent with those obtained by ion chromatography, a spectrophotometric assay and an ammonia-selective electrode [269]. 

A great advantage of spectrophotometric assays is that they can be carried out in microtiter plates or as filter paper assays, thus allowing a high sample throughput coupled with small sample volumes. Such systems were used for example for the screening of epoxide hydrolases [34]. A classical example of activity tests is for amylolytic enzymes where starch agar plates are stained with iodine after a certain reaction time. The radius of clear spots is a measure of the reaction rate [35]. 

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