Spectrophotometric assays for proteins

The measurement of protein concentration is an everyday occurrence in most laboratories working in the bimolecular sdences. An accurately determined protein concentration is an essential piece of information for many types of experiments that require a term for molarity of a protein, to monitor the various stages of a protein purification, to determine the amount of material to be added to an ELISA or coupled assay, or to normalize a number of different biological samples (e.g. number of cells or amount of cell extract). This type of assay must be straightforward and robust. It is also important that the assay can be used for a range of physically distinct protein preparations. For example, one 

If the material is contaminated vrith a UV-absorbing material whose spectrum is known, it may be possible to correct for the contaminant by measuring the absorbance at two wavelengths. A well-known example is protein contaminated by nucleic acids (or nucleotides), when the following equation can be used  

To this solution add 300 ml 10% (w/v) sodium hydroxide, mixing during the addition. 

This solution is stable for several months at room temperature. 

The sample should contain 0.5-4 mg protein in 0.2 ml water or buffer. 

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

Spectrophotometric assays for protein amino acid side chains... 

In this method the keyhole limpet haemoglobin conjugate was prepared as follows Keyhole limpet haemocyanin (KLH, Calbiochem, La Jolia, CA) and bovine serum albumin (BSA, BDH Chemicals) were coupled to the adduct (2), derived from 6-bromohexanoic acid and monoquat (3), via a carbodiimide reaction, as reported previously by Niewola et al. [184], The resulting conjugates contained 662mol of Paraquat per mole of KLH and 15mol of Paraquat per mole of 6-bromohexanoic acid. The amount of Paraquat bound to the protein was determined by spectrophotometric dithionite assay for Paraquat and the protein concentration was established by a standard Lowry test. 

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

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. 

Xylanase was assayed using birchwood xylan as substrate. The solution of xylan and the enzyme at appropriated dilution were incubated at 75°C for 3 min, and the reducing sugar was determined by the dinitrosali-cylic acid procedure (12) with xylose as standard. The released color development was measured spectrophotometrically at 540 nm. One unit of enzyme activity was defined as 1 pmol of reducing sugar released 1 min under the described assay conditions. Protein concentration was measured by the Lowry method (13) using bovine serum albumin as standard. 

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. 

Tris buffers Tris is also a much used buffer. However, it has one great disadvantage its pH is highly dependent on temperature and concentration. The pH of a Tris buffer will increase from 8.0 at 25 °C to 8.6 on cooling to 5 °C and on dilution of a 0.1 M solution at pH 8.0 to 0.01 M, the pH will fall to 7.9. This problem can only really be avoided by adjusting the pH of the buffer under the conditions of temperature and concentration where it is to be used. In addition, Tris has been shown, like phosphate discussed above, to interfere with many enzymic reactions, particularly those which have aldehyde intermediates. It also interferes with many chemical reactions, like the coupling of proteins to activated surfaces, and the Bradford assay for spectrophotometric determination of proteins. 

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. 

Assay procedures for glycosylated proteins incorporate varied methodologies (Al) and have been tailored to accommodate certain specific requirements. The identification and quantitation of the ketoamine link in a variety of proteins have rested mainly on the spectrophotometric thiobar-bituric acid assay. The standpoint is emphasized that short laboratory time assays for ketoamine-adducted proteins hold preeminence over rapid reporting of Hb Aj values as representing glycohemoglobin, unless strict attention to the control of sources of variation is adhered to. Exclusion of free glucose and the labile aldimine adduct is mandatory in all glycosylprotein assays. 

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

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). 

The proteins, avidin and streptavidin, are widely utilized in biotin analysis due to their outstanding aiSnity and specificity toward the binding of biotin (Zempleni et al. 2009). Generally an avidin-binding assay for the determination of biotin operates through the competition of sample biotin and labelled biotin (such as isotope-labelled biotin or biotinylated enzyme) with the limited number of avidin. Finally, the signals are acquired spectrophotometrically or electrochemically from the reaction of labelled enzyme with corresponding substrate or based on radioactivity counts. 

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