Comparative spectrophotometric assay

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. 

Spectrophotometric assays can be used for the estimation of the enantiosel-ectivity of enzymatic reactions. Reetz and coworkers tested 48 mutants of a lipase produced by epPCR on a standard 96-well microtiter plate by incubating them in parallel with the pure R- and S-configured enantiomers of the substrate (R/S-4-nitrophenol esters) [10]. The proceeding of the enzyme catalyzed cleavage of the ester substrate was followed by UV absorption at 410 nm. Both reaction rates are then compared to estimate the enantiomeric excess (ee-value). They tested 1000 mutants in a first run, selecting 12 of them for development of a second generation. In this way they were able to increase the enantiomeric excess from 2% for the first mutants to 88% after four rounds of evolutive optimization. 

In most studies of DPO activity, the main objective is usually a simple comparison of the potential of a particular tissue to undergo enzyme-catalyzed browning, for example, a comparison of the potential for enzymic browning of different apple or mushroom cultivars. Related to this are comparative studies of different inhibitors and processing regimes to control enzymic browning. In these circumstances, it is usually sufficient to provide comparative measurements rather than absolute values of enzyme activity, in which case results can be expressed in arbitrary units such as AmV/min for 02 electrode assays or AA/min for spectrophotometric assays. If more precise units are required, the 02 electrode results should be expressed as Anmol 02/min/(j.g protein. 

In practice it is often more convenient to measure the release of a phenol from an aryl phosphomonoester. Standard serum phosphatase methods employ phenyl phosphate (188), p-nitrophenyl phosphate (189), phenolphthalein monophosphate (140), or thymolphthalein monophosphate (141) where the phenol released can be determined spectrophoto-metrically [only the Bodansky method (13) uses a Pi determination]. A number of fluorogenic substrates have been used for phosphatase studies, e.g., jS-naphthyl phosphate (30, 148), 4-methylumbelliferyl phosphate (143), and 3-O-methylfluorescein phosphate (144) The main advantage here is the much greater sensitivity of fluorescence as compared with spectrophotometric assays as little as 1 pmole of 4-methyl-umbelliferone can be detected in continuous assay. 

G8. Girotti, S., Ferri, E., Maccagnani, L., Budini, R., and Bianchi, G., Plasma antioxidant capacity determination Comparative evaluation of chemiluminescent and spectrophotometric assays. Talanta 56, 407-414 (2002). 

Since, In practice, the efficiency frequently varies, it is necesseuy to determine the counting efficiency before one can compare different samples. A radioactive standard of acciirately known activity is essential for the determination of the efficiency counting. The use of radioactive standards is in principle similar to that of standards in colorimetric or spectrophotometric assays. There are two types of standards (i) internal standard (this is usually a p-emitter of accurately known activity which, when dissolved in unquenched scintillation mixture, provides a reference standard), and (ii) external standard (ay-emitter incorporated in most instruments). 

As a practical matter, it often turns out that the amount of the enzyme in the active form is the factor most susceptible to change ki, ki, Xo, and Oo are comparatively more stable. Not only is the enz3une labile and convertible into an inactive form that will not combine with the substrate, but the active enzyme-substrate complex is readily converted into an inactive complex. We have found that the direct spectrophotometric assay of the amount of active enzyme-substrate complex is the most effective, and sometimes is the only, satisfactory method for the measurement of enzyme activity under unfavorable chemical or physical conditions (13). 

Identification of dyes on dyed textiles is traditionally carried out by destructive techniques [493], TLC is an outstanding technique for identification of extracted dyestuffs and examination of inks. Figure 4.9 shows HPTLC/SERRS analysis of acridine orange [492], Wright et al. [494] have described a simple and rapid TLC-videodensitometric method for in situ quantification of lower halogenated subsidiary colours (LHSC) in multiple dye samples. The results obtained by this method were compared with those obtained by an indirect TLC-spectrophotometric method and those from HPLC. The total time for the TLC-videodensitometric assay of five standards and four samples applied to each plate was less than 45 min. The method is applicable for use in routine batch-certification analysis. Loger et al. [495,496] have chromatographed 19 basic dyes for PAN fibres on alumina on thin-layer with ethanol-water (5 2) and another 11 dyes on silica gel G with pyridine-water... 

An ultraviolet spectrophotometric analysis is used to assay pseudoephedrine hydrochloride in tablets. A portion of finely powdered tablets equivalent to approximately 30 mg of pseudoephedrine hydrochloride is placed in a distilling flask which is part of a micro-steam distillation apparatus. Sodium chloride, water, and concentrated sodium hydroxide are added. A minimum of 30 ml of distillate is collected in a volumetric flask containing dilute hydrochloric acid. The flask is made to volume with distilled water and the absorbance of the solution is determined at 257 nm in 1 cm cells and compared to a solution of known concentration of NF Pseudoephedrine Hydrochloride Reference Standard.1... 

The suggested derivative spectrophotometric method and PLS-1 were applied to determirration of caffeine in energy drirrks (Table 31.2). The assay results obtained by both methods were statistically compared at the 5% level. As shown in Table 31.2 there was no sigrrifrcant differences between the mean values and precisiorrs of two methods. 

Biochemical research often requires the quantitative measurement of protein concentrations in solutions. Several techniques have been developed however, most have limitations because either they are not sensitive enough or they are based on reactions with specific amino acids in the protein. Since the amino acid content varies from protein to protein, no single assay will be suitable for all proteins. In this section we discuss five assays three older, classical methods that are occasionally used today and two newer methods that are widely used. In four of the methods, chemical reagents are added to protein solutions to develop a color whose intensity is measured in a spectrophotometer. A standard protein of known concentration is also treated with the same reagents and a calibration curve is constructed. The other assay relies on a direct spectrophotometric measurement. None of the methods is perfect because each is dependent on the amino acid content of the protein. However, each will provide a satisfactory result if the proper experimental conditions are used and/or a suitable standard protein is chosen. Other important factors in method selection include the sensitivity and accuracy desired, the presence of interfering substances, and the time available for the assay. The various methods are compared in Table 2.3. 

The greatest advantage of the spectrophotometric method is that it is direct and rapid, requires no sample workup, and allows for continuous assays of lipase activity compared to the multiple fixed-time-point analyses incumbent within Basic Protocols 1 and 2. The spectrophotometric method can also be done using very small volumes (as small as 1 ml) and is suitable for following the course of purification (such as in chromatographic fractions) or adaptable to 96-well plates (and subject to automation, if available). Thus, it is the method of choice for screening several samples or preparations for lipase (esterase) activity. 

The other major types of assay are all spectrophotometric (Alternate Protocols 1 and 2) and usually involve measuring the production of the highly colored end-product of the DPO reaction. They are used widely because they are rapid, can be carried out with the simplest of colorimeters/spectrophotometers, and can be adapted to use microtiter plates (Alternate Protocol 3). Thus they are ideal for simple routine comparative or monitoring studies. A major weakness of these colorimetric procedures is that they measure the end-product of a complex sequence of reactions, and different substrates yield different... 

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