Proteins spectrophotometric assay

Reznick A, Packer L (1994) Oxidative damage to proteins spectrophotometric method for carbonyl assay. Methods Enzymol 233 357-363... 

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 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. 

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. 

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. 

After 1 min, add 0.1 mL of 10% aqueous sodium chloride to each tube. Where there is excess protein in the tubes, the sol will not change color, but in those tubes where there is insufficient protein to stabilize the gold, flocculation will have occurred and the liquid will be blue. The correct concentration of protein is the minimal amount that will inhibit flocculation. Horisberger (10) suggests that for accurate determination of color change, a spectrophotometric assay should be used. 

Lead Determine as directed for Method I in the Atomic Absorption Spectrophotometric Graphite Furnace Method under Lead Limit Test, Appendix MB, using a 1-g sample. Loss on Drying Determine as directed under Loss on Drying, Appendix EC, drying a 2-g sample at 105° for 2 h. Starch The remainder, after subtracting from 100.0% the sum of the percents of Ash (Total), Loss on Drying, and Protein (see Assay, above), represents the percent of starch in the sample. 

One milligram of microsomal protein is added to 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaF, 10 mM dithiothreitol, 1 mM EDTA, 20% glycerol (v/v), 150 iM 5-cholestene-3/3, 7a-diol, and 0.915% CHAPS. The reaction is initiated by 1 mM NAD+ to give a final reaction volume of 1.0 mL. After incubation at 37°C for 5 minutes, the reaction is terminated by adding 2 mL of 95% ethanol. An internal recovery standard, 4-cholesten-3-one (3 fig in methanol) is also added. The steroid products are extracted into 5 mL of petroleum ether (repeated twice). After the ether has been removed at 40°C under a stream of nitrogen, the products are dissolved in 100 fxL of mobile phase and 20 ju.L is injected into the column. The amount of product formed is linear with protein (to 1.5 mg) and with time (up to 10 min, 1 mg protein). The assay is much more sensitive than the direct spectrophotometric assay, and it avoids the use of thin-layer chromatography and radioisotopes described in other methods. 

The enzyme activity was measured by a continuous spectrophotometric assay (see Methods), active site concentration was determined by FAD absorption at 452 nm (8 = 12.83 mM-icm-i) as described by Frederick et al. (1990) and the protein concentration was measured by the Bradford assay (BioRad reagent) using bovine serum albumin as standard, or by its absorption at 280 nm using a published factor of 1.67 O.D. per mg (Swoboda Massey, 1965). The specific activity was 430 U/mg, and the overdl yield of enzyme, based on active sites measurement (452 nm absorption), was about 40%. 

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. 

Cheng Q, Wang ZX, Killilea SD (1995) A continuous spectrophotometric assay for protein phosphatases. Anal Biochem 226 68-73... 

The presence of copper in erythrocuprein was first demonstrated by Mann and Keilin (55). In 1970 zinc was found in human erythrocuprein (69). The metal ions were measured by different spectrophotometric assay procedures using 2,2-biquinoline, bis cyclohexanone oxalyldihy-drazone, and dithizone as chelating ligands (85, 97—100). Alternatively, atomic absorption spectroscopy (86), neutron activation analyses, and emission spectroscopy (<5< ) were successfully employed. From the neutron activation analyses it became apparent that metals other than zinc and copper were present in amounts less than 0.1 g-atom per 33,000 g of protein. From the different analyses (Table 3) it can be concluded that erythrocuprein contains 2 g-atoms of each of copper and zinc. 

R6. Resnick, A. Z., and Packer, L., Oxidative damage to proteins Spectrophotometric method for carbonyl assay. Meth. Enzymol. 233, 357-363 (1994). 

Spectrophotometric assay of P700 and light-induced absorption changes were carried out with a Hitachi 556 dual wavelength spectrophotometer(1,2). EPR measurements of Fe-S proteins were performed as described previously(2). 

Spectrophotometric assays for protein amino acid side chains... 

Figure 1 Presqualene and squalene synftiesis. A, Relative PS and S activities measured by spectrophotometric assay during the incubation of M105 and SI05 extracts from WS, RS and MS yeasts. B, PS /S ratios. The enzyme activity was expressed as A OD / min / mg protein.

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