Contents Spectrophotometric assay

Several useful methods for the quantitative determination of protein solutions were discussed in Chapter 2. Two of those methods, the Bradford protein assay and the direct spectrophotometric assay, will be applied to the a-lactalbumin solutions. Neither of these assays is specific for a certain type of protein rather they both estimate total protein content. 

Shinkuma et al. [14] reported a spectrophotometric assay of different commercial mefenamic acid capsules. The contents of 20 capsules of each brand were carefully removed and accurately weighed. After finding the mean weight of the capsule contents, the contents were mixed uniformly and used as the brand sample. A weighed amount of this powder, equivalent to about 150 mg of mefenamic acid, was dissolved in 200 mL... 

Corn seeds were germinated between moist paper towels and were then treated with 5 uM CDAA either 1 or 2.5 days prior to assay for GSH af 6 days after planting (Table V). Using the reagent DTNB [2,2-dithiobis (2-nitrobenzoic acid)], root GSH contents were assayed spectrophotometrical ly as previously described (20). 

Assay In the case of herbal preparations with constituents of known therapeutic activity, assays of their content are required with details of the analytical procedure. Wherever possible, a specific, stability-indicating procedure should be included to determine the content of the herbal substance in the herbal preparation. In cases where use of a nonspecific assay is justified, other supporting analytical procedures should be used to achieve overall specificity. For example, where a UV/VIS spectrophotometric assay is used for anthraquinone glycosides, a combination of the assay and a suitable test for identification (e.g., fingerprint chromatography) can be used. In the case of herbal preparations where the constituents responsible for the therapeutic activity are not known, assays of marker substances or other justified determinations are required. The appropriateness of the choice of marker substance should be justified. 

The molar ratios of the amino-acid residues were determined after erythrocuprein was subjected to acid hydrolysis at 110°C for 20 to 96 hours. The method of Moore and Stein was used throughout (88, 89). The sulfhydryl content of human erythrocuprein was determined using the spectrophotometric assay of Boyer (90). Total half-cystine was analysed as S-carboxy methyl cysteine in acid hydrolysates (91) of reduced and alkylated erythrocuprein, or as cysteic acid in acid hydrolysates oxidized with performic acid. The data are summarized in Table 2. 

Cost-effective flow injection spectrophotometric assay of iron content in pharmaceutical preparations using salicylate reagent. Talanta, 64,1237-1240. 

All fractions were analysed for AE activity and protein content. The protein content was measured spectrophotometrically according to Bradford using the BioRad protein assay kit with Y-globulin as standard (5). 

The British P 1993 [15] describes a procedure to determine benzoic acid in benzoic acid ointment that also contains salicylic acid. In this method, 2 g of the ointment is added to 150 mL water, and warmed until melted. The solution is titrated with 0.1 M NaOH, using phenolphthalein solution as the indicator. Salicylic acid is assayed by a spectrophotometric procedure, and its content subtracted from the total acid value to obtain the benzoic acid content. 

The United States Pharmacopoeia 23 [11] and Indonesian Pharmacopoeia IV [9] describe the assay of benzoic acid and salicylic acid in ointments. Two chromatographic columns (20 x 2.5 cm) are used to effect the separation. One transfers a mixture of 1 g siliceous earth and 0.5 mL diluted phosphoric acid (3 in 10) to the first column (A), then packs above this a mixture of 4 g siliceous earth and ferric chloride-urea reagent. A mixture of 4 g siliceous earth and 2mL of sodium bicarbonate solution (1 in 12) is packed into the second column (B). For analysis, column A is mounted directly above column B. The sample solution is inserted onto column A, allowed to pass into the column, and then washed with 2-40 mL of chloroform. Benzoic acid can be eluted from column B by using a 3 in 100 solution of glacial acetic acid in chloroform. The benzoic acid content then can be determined by a spectrophotometric method such as that described earlier (section 4.5). 

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. 

Describes a simple and sensitive spectrophotometric method to estimate the content of total carbonyl compounds in rancid fats and foods by trapping them with 2,4-DNPH the technique determines total carbonyls, including those that are nonvolatile, decreasing the ability of the assay to correlate well with sensory data. Although gas chromatographic techniques are better suited for determining volatile carbonyl compounds from lipid oxidation, this is still the classical colorimetric assay. 

Con A Binding Assay. For each binding experiment a number of 60 by 15 mm Petri dishes containing 3.0 ml PBS and 1.5 ml of a 3 mg/ml Con A solution were adjusted to the required conditions of temperature or pH. A volume of 0.6 ml of a well-mixed 1 1 (v/v) suspension of dextran gel spheres was added with a 1 ml plastic pipet to each dish, followed by incubation with shaking. Control samples contained methyl a-D- glucopyranoside (a-MG) at a final concentration of 0.1 M. At various intervals the contents of a sample and a control dish were centrifuged quickly and the supernatant withdrawn for UV spectrophotometric analysis at 280 nm... 

For example, when a heart attack occurs, a lack of blood supplied to the heart muscle causes some of the heart muscle cells to die. These cells release their contents, including their enzymes, into the bloodstream. Simple tests can be done to measure the amounts of certain enzymes in the blood. Such tests, called enzyme assays, are very precise and specific because they are based on the specificity of the enzyme-substrate complex. If you wish to test for the enzyme lactate dehydrogenase (LDH), you need only to add the appropriate substrate, in this case pyruvate and NADH. The reaction that occurs is the oxidation of NADH to NAD+ and the reduction of pyruvate to lactate. To measure the rate of the chemical reaction, one can measure the disappearance of the substrate or the accumulation of one of the products. In the case of LDH, spectrophotometric methods (based on the light-absorbing properties of a substrate or product) are available to measure the rate of production of NAD+. The choice of substrate determines what enz)rme activity is to be measured. 

Z. Feher, G. Horvai, G. Nagy, Z. Niegreisz, K. Toth, and E. Pungor, A Polarographic and Spectrophotometric Routine Analyzer for Assaying Content Uniformity in Pharmaceutical Quality Control. Anal. Chim. Acta, 145 (1983) 41. 

As described for piperazine. Solution I contains 3,0492 g ethanbutol dihydrochloride in 1000 ml water, acidified with 1 ml HCl (37%). 10 samples are withdrawn between 0,5 and 60 min. The content of dinitroso-ethairbutol is assayed spectrophotometrically at 240 nm. 

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). 

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