Protein, estimation methods

It is very often observed that during a purification process the differences increase between the real amounts of a protein and the values obtained by any method, e.g., total enzyme activity, because the measured signal produced by a protein mixture differs from that of a pure protein. Furthermore, the amount of a given protein determined by a distinct protocol differs from the expected amount by portioning, as shown in Table 1.1. To avoid additional mistakes with the already uncertain process, the protein estimation method should not be changed during a purification process. 

Protein estimation Proteins were determined by the method of Lx)wry et al. (1951), using bovine serum albumin as standard. 

The aromatic rings in the protein absorb ultraviolet light at an absorbance maximum of 280 nm, whereas the peptide bonds absorb at around 205 nm. The unique absorbance property of proteins could be used to estimate the level of proteins. These methods are fairly accurate with the ranges from 20 p,g to 3 mg for absorbance at 280 nm, as compared with 1 to 100 p,g for 205 nm. The assay is non-destructive as the protein in most cases is not consumed and can be recovered. Secondary, tertiary and quaternary structures all affect absorbance therefore, factors such as pH, ionic strength, etc can alter the absorbance spectrum. This assay depends on the presence of a mino acids which absorb UV light (mainly tryptophan, but to a lesser extent also tyrosine). Small peptides that do not contain such a mino acids cannot be measured easily by UV. 

The Kjeldahl total nitrogen determination method is not very sensitive, hut it suits well for analyzing insoluble samples without preceding disintegration. Automated Kjeldahl protein estimations are used especially in food analysis. 

Students will isolate intact mitochondria from beef heart and fractionate them to prepare submitochondrial particles. Each fraction will be characterized by protein estimation by the biuret method and measurement of malate dehydrogenase and monoamine oxidase activity. 

The selection of a protein standard is potentially the greatest source of error in any protein assay. Of course, the best choice for a standard is a highly purified version of the predominate protein found in the samples. This is not always possible nor always necessary. In some cases, all that is needed is a rough estimate of the total protein concentration in the sample. For example, in the early stages of purifying a protein, identifying which fractions contain the most protein may be all that is required. If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve with the selected protein assay method. 

For greatest accuracy of the estimates of the total protein concentration in unknown samples, it is essential to include a standard curve in each run. This is particularly true for the protein assay methods that produce nonlinear standard curves (e.g., Lowry method, Coomassie dye-binding method). The decision about the number of standards used to define the standard curve and the number of replicates to be done on each standard depends upon the degree of nonlinearity in the standard curve and the degree of accuracy required of the results. In general, fewer points are needed to construct a standard curve if the color response curve is linear. For assays done in test tubes, duplicates are sufficient however, triplicates are recommended for assays performed in microtiter plates due to the increased error associated with microtiter plates and microtiter plate readers. 

The protein-to-protein variation observed with the various protein assay methods makes it obvious why the largest source of error for protein assays is the choice of protein for the standard curve. If the sample contained IgG as the major protein and BSA was used for the standard curve, the estimated total protein concentration of the sample will be inaccurate. Whether the concentration was underestimated or overestimated depends upon which total protein assay method was used. If the Coomassie... 

Plus Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be underestimated by -40%. (From Table B1.1. 5, the response ratio for IgG is -0.58 for IgG compared to 1.00 for BSA.) If the BCA Protein Assay Reagent was used, the total protein (IgG) concentration in the sample would be overestimated by -15%. (From Table B1.1.5, the response ratio for IgG is -1.15 for IgG compared to 1.00 for BSA.) On the other hand, if BGG had been used for both standard curves, the total protein estimates for the sample would have been in much greater agreement between the two methods. 

The amount of time required to complete a total protein assay will vary for the four colorimetric total protein assay methods described. For the puipose of providing an estimate of the amount of time required to perform a run by each method, it was assumed that the run included twenty samples and eight standards (including the blank) and that each sample or standard was assayed in duplicate using the standard tube protocol. The estimates do not include the time spent obtaining the samples or the time it takes to prepare the samples for... 

Hageman et al. [3.13] calculated the absorption isotherms for recombinant bovine somatotropin (rbSt) and found 5-8 g of water in 100 g of protein, which was not only on the surface but also inside the protein molecule. Costantino et al. [3.72] estimated the water monolayer M0 (g/100 g dry protein) for various pharmaceutical proteins and for their combination with 50 wt% trehalose or mannitol as excipient. They compared three methods of calculating MQ (1) theoretical (th) from the strongly water binding residues, (2) from conventional adsorption isotherm measurements (ai) and (3) from gravimetric sorption analysis (gsa) performed with a microbalance in a humidity-controlled atmosphere. Table 3.5 summarizes the results for three proteins. The methods described can be helpful for evaluating RM data in protein formulations. 

A useful procedure for estimating adenylate cyclase in intact cells and tissues is to incubate the tissue with labelled adenine and then measure the rate of labelling of cyclic AMP [112]. Adenine readily penetrates cells and is partially converted to ATP. In heart slices, the ATP newly synthesised from radioactive adenine was found in equilibrium with the existing pool used for the production of cyclic AMP the specific activity of the newly-formed cyclic AMP was similar in the presence and in the absence of stimulatory hormone [113]. The prelabelling method has been compared with the protein-binding method in brain slices [114,115]. Increases in total levels of cyclic AMP and increases in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides labelled by prior incubation with radioactive adenine occurred on similar time courses and to similar extents. Radioactive cyclic AMP represented a small (7-13%) but relatively constant fraction of the total amount of cyclic AMP. These results provided no evidence for the presence of more than one major compartment of adenine nucleotides in brain slices that serve as a source of nucleotide precursor for cyclic AMP. The nucleotides of this compartment were uniformly labelled by incubation with radioactive adenine [116]. 

Figure 1.5. Relationship between actual and measured BSA concentration in six samples, showing that this method produces comparable results to the Bradford and BCA total protein assays. [Reprinted, with permission, from K. C. Bible, S. A. Boemer, and S. H. Kaufmann, Anal. Biochem. 267, 1999, 217-221. A One-Step Method for Protein Estimation in Biological Samples Nitration of Tyrosine in Nitric Acid. Copyright 1999 by Academic Press.]...

Figure 24-5 Frequency distribution of quantitative results combined with returns of "nil, zero, or not detected for distributions of salt solution and normal urine. Quality control of total protein measurement demonstrates the widespread variation in results achieved using different methods, (from Chombers RE, Bullock DG, Wh/cfier JT. Urinary total protein estimation Fact or fiction Nephron 1989 53 33. Reproduced with permission of S. Karger AG, Basel, Switzerland.)...

Free or unbound cortisol represents the biologically active form of the circulating hormone, and its concentration is practically independent of alterations of its transport proteins. Various methods have been developed for estimating the free fraction in serum, but these assays are technically demanding, expensive, and not in general use. The measurement of urine free cortisol comes closest to providing an estimate of the free hormone concentration. As mentioned previously, approximately 2% of cortisol is excreted into the urine in a free form, and its measurement has been shown to be of use as a screening test for cortisol hypersecretion. However, P-hydroxycortisol has been reported to interfere with the immunoassay of free cortisol in urine. ... 

Direct two-step and one-step immunoassays estimate free hormone concentrations by using antibody extraction techniques. Although it is often clauned or implied that these immunoextraction assays measure FT or FT3 directly, virtually all methods do so indirectly by relating test results to extracted serum calibrators with free hormone values that have been independently measured using reference methods (e.g.,. direct equilibrium dialysis/RlA). Further, studies have shown that all of the free hormone estimate methods on current instrument platforms are binding protein dependent to some extent. ... 

R20. Rogers, J. A., and Watson, D., Evaluation of the specific gravity drop method for serum protein estimation. Med. J. Australia 62, 690-694 (1963). 

Curve-titting techniques, such as those described by Coakley and James, may be employed tor the analysis ot the relationship between silver stain densities and protein concentrations. Coakley and James developed these techniques to examine the similar curvilinear relationships which are tound in the Folin-Lowry method ot protein estimation (7J ). With caretul measurement ot total stain densities, estimates ot relative protein concentrations have been made over a 220 told concentration range with six puritied proteins(3 ). 

Fleurence, 1999b). For example, McDermid and Stuercke (2003) used Lowry s method (Lowry et ah, 1951), which is specific for protein. Another method used for the estimation of protein is Biuret method (Manivannan et ah, 2008). Gressler et ah (2011) reported that the value of soluble protein obtained by Bradford s method (with bovine serum albumin as a standard) was quite similar to that determined by the method based on the nitrogen-to-protein factor 4.43. 

Like ionic liquids, many chemical products are designed using property estimation methods, as discussed in the next section. These include polymer membranes and refrigerants. However, for pharmaceuticals, the properties of proteins are normally not estimated. Rather, they are determined experimentally in the laboratory as discussed next. 

As mentioned before, the amount of soluble tannin that causes astringency in persimmon fruits is usually estimated visually by the tannin print method and can be measured quantitatively by the Folin-Denis method. There is also a protein precipitation method for the measurement of soluble tannins (Hagerman and Butler 1978). In that method, the soluble tannin content is assayed by the addition of the sample to a standard solution of protein and the isolation of insoluble tannin-protein complexes. The complexes are dissolved in alkaline solution, to which ferric chloride is added. The absorbance of the solution at 510 nm is measured. 

Quantitation. Various standard protein analytical methods can be nsed for quantitation of collagen. These include determination of the total nitrogen content by Kjeldahl analysis, followed by estimation of the collagen concentration... 

Protein mass is also estimated empirically from a large number and variety of nephelometric, turbidimetric and protein volume methods. In general, these have the advantage of being applicable to smaller samples, and being faster for routine analyses. What apparently has not always been fully appreciated is that a complete empiricism and a whole new set of possible errors are introduced with these methods. In general they... 

Abstract— This paper reported an improvement to the previously proposed maximum-minimnm variation (MMV) test for protein estimation. The proposed method ntilizes single protein test as compared to three protein tests for MMV. In addition, a new computerized algorithm namely maximum-minimum area variation (MMAV) has been proposed in order to estimate the protein concentration in the latex glove. The new proposed technique, give significantly better results in terms of consistency and accuracy than existing methods. Besides, the method reduces the chemicals usage, lab consumables, and the need for certain hardwares. Thus, it is more environmental friendly. 

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