The 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. 

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

Among chromenes, only the spiropyrans and their heterocyclic derivatives have found a wide practical application as photochromic substances.5 6,7-Chromenediols have been proposed as analytical reagents for the spectrophotometric assay of rare earth cations.279 Chromenes with structure and activity similar to those of hashish constituents have been prepared.280 A number of chromenes, mostly with aryl substituents in positions 2,3, and 4 have been patented as biologically active substances.126,280 290... 

Although the spectrophotometric assay of proteins is fast, relatively sensitive, and requires only a small sample size, it is still only an estimate of protein concentration. It has certain advantages over the colorimetric assays in that most buffers and ammonium sulfate do not interfere and the procedure is nondestructive to protein samples. The spectrophotometric assay is particularly suited to the rapid measurement of protein elution from a chromatography column, where only protein concentration changes are required. 

B 7. A solution of purified DNA gave in the spectrophotometric assay an A16a of 0.35 when measured in a 1-cm quartz cuvette. What is the concentration of the DNA in /xg/mL ... 

B 9. What amino acid residues are detected when the spectrophotometric assay is used to quantify proteins Are those amino acids present in the same quantity in all proteins Explain how this may affect measurement of proteins by this method. 

Assays for Antioxidative Activity Spectrophotometric and polarographic (9) assays were utilized to monitor levels of antioxidative activity. In the spectrophotometric assay, a 2-ml portion of linoleic acid emulsion (9) was placed in a test tube along with 10 pi of a sample to be assayed, and from this, 200 pi were withdrawn and mixed with 2 ml of 100% methanol and 6 ml of 60% methanol-water. The remainder of the 2 ml of emulsion and sample mixture was then incubated at 37°C for 15 to 20 h. Meanwhile, the absorbance of the methanol solution to which the 200 pi of sample had been added was measured at 234 nm. After incubation, the absorbance of 200 pi of the emulsion and sample mixture was also measured at 234 nm in the same manner. Antioxidative activity (A.O.A.) was calculated using the equation ... 

For a more quantitative approach, HPLC detection is recommended (Halliwell, 1988). However, the spectrophotometric assay described here is certainly ideal for qualitative assessment of hydroxyl radical production. 

In each case make up the indicated solution and then pick up the standard assay procedure at step 3. The results of blank A2 should have shown that, in the absence of glucose and fmctose, the mixture of reagents used in the standardization runs will yield no significant color. The absorbances obtained in mns B1 to B5 can thus be attributed to the action of the reagent on the GFS. These data will be used to constmct a calibration curve for the spectrophotometric assay. 

K4. Karmen, A., A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood serum. ]. Clin. Invest. 34, 131 (1955). 

As indicated in Fig. 7.5e, the spectrophotometric assay revealed the inhibition of the unphosphorylated Brc-Abl by the wrapper of the 249-252 and 300-316 dehydrons (the I- and II-methylation product) improved over Gleevec levels. Furthermore, the inhibitory impact of the dehydron wrapper became selective for Brc-Abl vis-a-vis C-Kit and Lck. Dehydrons 249-252 and 300-316 are absent in the latter kinases and, consistently, the drug designed to better wrap them has very low inhibitory impact against C-Kit and Lck. 

Until recently, there were two methods for analysis of TP activity spectrophotometry and TFC, both of which were based on the conversion of thymidine to thymine. The spectrophotometric assay is based on the difference in molar-extinction coefficients between thymidine and thymine at alkaline pH. A fixed time-point at 90 min (12) or 16 h (13) has been used. 

The second method uses the conversion of [14C]-thymidine to [14C]-thymine or vice versa as separated by TFC. Unlike the spectrophotometric assay, data on linearity with time and protein concentration is available for the TFC assay (14). The main disadvantage of this particular method is the limitation of the number of samples that can be analyzed at one time owing to the capacity of the available chromatography equipment. 

The spectrophotometric assay is based on a POase indicator reaction to measure the amount of H2O2 liberated. For this reason an excess of POase and H-donor (Section 10.2.1.4.2) has to be used. One unit (U) is then expressed as the amount of enzyme liberating 1 pmole of H2O2 per min at 25°C. 

In the spectrophotometric assay system (Bergmeyer et al., 1974 Table 10.17), the total reaction mixture (3 ml) should contain about 0.5 U GOase (about 2 pg active enzyme) per ml (at 25°C), added... 

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. 

Several workers " studied the polarographic behaviour of nitrofurans. Nitrofurazone in animal feed has been quantitatively assayed by polaro-graphy. Polarographic assay of nitrofurans will require more basic research before it can be used on a large scale. However, the spectrophotometric assay of nitrofurantoin has been replaced by a polarographic assay in the first U.S. P. XVI supplement (U.S. Pharmacopoeia Convention, 1962). 

The measurement of oxygen uptake provides a direct index of metabolic rate this measurement can be done simply by placing experimental animals In a calibrated vessel maintained at constant temperature and connected to an oxygen analyzer (86). Repeatedly used as an Index of thyromimetic activity have been the in vitro assay of oxygen uptake by suspended mitochondria and the spectrophotometric assays of rat liver mitochondrial a-glycerophosphate dehydrogenase (87) and of cytoplasmic malic enzyme (88). 

The peroxide method has proven to be the most useful for this purpose, owing to the high acidity of the medium in which the reaction is conducted. Interferences are observed only in the presence of V, Mo, or F, but these species are not normally present in U.S.P. grade titanium dioxide. In the spectrophotometric assay method, the absorption maximum at 410 nm is used to determine the titanium concentration after the oxide is dissolved [39]. The spectrophotometric endpoint of the peroxide method has been combined with flow injection analysis techniques to yield an automated procedure [40]. 

Rubisco activity was measured by radiochemical method and enzyme quantity by immunorocket as in (3). Extracts for Sucrose-P-synthase were desalted on G-25 Sephadex column and the reaction was run as in (3), except that UDP glucose and Fructose 6 P were adjusted to 25 and 10 mM respectively.The spectrophotometric assay was used to measure ADPgIucose pyrophosphorylase (3). Sucrose synthase and neutral invertase were assayed as in (4). 

The curve in Figure 8 is the equation and the points are the data derived from the reduction of the data in the inset of the figure. The activity constants derived from the Michaelis-Menten equation are Vmax = 0.11 nmol-min and Km = 0.1 mM. The spectrophotometric assay values of GUS in solution are Vmax == 1.55 nmohmin, and Km = 0.078 mM. The velocity maximum value for the immobilized enzyme is about 1/10 the value of the solution assay value (/P), probably due to the immobilization in the PSi surface framework restricting access to the enzymatic sites, effectively reducing the concentration of the available enzyme. The Km values, however, are similar, indicating the activity of the enzyme is not affected by the immobilization. 

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