Assay for protein content

A number of methods  are  available  for  measuring protein concentration, each being based on a specific property  of proteins, and each having certain advantages  and disadvantages.  Consequently, the different methods are more  or  less suitable for different  applications  and it is useful to have insight into these methods so that one can decide which one to use for a given application.

1. Absorption of ultraviolet light

UV-absorption is perhaps the  most simple method  for measuring the concentration of proteins in solution.  A typical protein absorption spectrum has an absorption  peak  at 280 nm,  due to  the  aromatic  amino acids, such as tryptophan  and tyrosine.  Below 220 nm  the  absorption also increases strongly, due to  peptide  bonds, which absorb maximally  at 185 nm.  The  extinction  coefficients  of different  proteins  tend  to  be different at 280 nm, due to their different aromatic  amino  acid contents, while below 220 nm  the  extinction  coefficients  are  more  similar.  It  is difficult to  measure absorption  accurately  in  this  part  of the  spectrum, however, partly because oxygen forms begins to absorb in this region.

Because the  extinction  coefficients  of proteins  differ,  UV-absorption is useful as a qualitative  measure,  for detecting  the  presence  of protein, but is less useful  for  accurate  quantitative  measurements,  except  for  pure proteins  of known  extinction  coefficient.  Because of its  simplicity,  UV- absorption is the method favoured for continuous (semi-quantitative) monitoring of the protein concentration in the eluate from chromatography columns.

One of the limitations of UV-absorbance, as a method  for measuring protein, is that  UV-absorbing, non-protein, compounds may interfere with the measurement.  Nucleic acids, which are ubiquitously present in biological material,  absorb UV radiation  strongly,  with a profile overlapping that of protein, but with a maximum at 260 nm.  An elegant

method for eliminating the absorption  due to  nucleic acids, thus allowing a measurement  of protein  in the  presence  of nucleic acid, has  been proposed by Groves et al.⁸.

In measuring the  concentration  of proteins  by their  UV-absorbance, remember that  the  extinction  coefficient  (or  absorption  coefficient)  is given by the equation:-

where, A = absorbance

am =  molar extinction coefficient

c = molar concentration of protein in solution

= length of the light path through the solution (usually 1 cm).

If the concentration is given in g/litre, then the equation becomes:-

Where as= specific extinction coefficient

Note that a_m = a_s x MW.

2. The biuret assay

In alkaline solution, proteinsreduce cupric (Cu²⁺) ions tocuprous (Cu¹⁺) ions which react with peptide bonds to give a blue coloured complex. This reaction is called the biuret reaction and is named after the compound biuret (I), which is thesimplestcompound thatyields the

characteristic colour.

Because the reaction is with peptidebonds, thereis littlevariationin

the colour intensity given by different proteins.Thebiuret method can be used for the measurement of protein concentrationin thepresenceof

polyethylene glycol, a common protein precipitant. A disadvantage of the biuret method is thatitis relativelyinsensitive, so thatlarge amountsof proteinare required for theassay.Amore sensitive variant of the method, the micro-biuret assay, has been devised, which overcomesthis limitation tosome extent.Another limitation is that amino buffers, such as Tris, which are commonly used in the pH range ca. 8-10, can interfere with the reaction.

3. The Lowry assay

The Lowry assay may be considered as anextensionof thebiuret

assay. Initially, a copper-protein complex is formed, as in the biuret assay.The cuprous ions then reduce the so-called Folin-Ciocalteu reagent, a phosphomolybdic-phosphotungstate complex, to yield an intense blue colour.An advantageof theLowry over thebiuret assay is that it is much moresensitive, and thus consumes much less of the protein sample.A disadvantage of theLowry assay is thatitis more sensitive to interference,a consequence of themorecomplicated chemistry involved.The Lowry assay has been reviewed by Peterson.

4. The bicinchoninic acid assay

Another development of the biuret reactionis the bicinchoninic acid (BCA) assay.Bicinchoninic acidformsa 2:1complexwith cuprous ions formed in the biuret reaction, resulting in a stable, highly coloured chromophore with an absorbance maximum at 562 nm^13,14. The BCA assay is more sensitive than the Lowry methodand is also less subject to

interference by a number of commonly encountered substances.As the reaction is dependent, in thefirst instance, on thereduction of cupric ions tocuprous ions by theprotein,it is sensitivetointerferenceby

strong reducing agents, e.g. ascorbic acid.Thislimitationalsoappliesto the biuret and Lowry assays.

5. The Bradford assay

A proteinassay which is rapidly becoming themostcommonly used method, due to its simplicity, sensitivity and resistance to interference,is

the dye-binding method described by Bradford. Coomassie blue G-250,

dissolved in acid solution, below pH 1, is a red-brown colour but regains its characteristic blue colour when it becomes bound toa protein.The

concentration of protein can therefore be measured by theextentto which theblue colour,measured at595nm, is restored.Coomassie blue G-250 binds largely to basic and aromatic amino acids.Different proteins will differ in their content of these amino acids and so,ideally, a standard curve should be elaborated for eachspecific protein.A modificationhas been introduced by Read and Northcote¹⁶ to overcome this problem to some extent.Adisadvantageof theBradfordassayisthatthereagent tends tostick to glass and plastic ware.For thisreason,theuse of disposable cuvettes is recommended although, if necessary, the dye can be removed from surfaces by using SDS.

References

-Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .

Groves, W. E., Davis, F. C. and Sells, B. H. (1968) Spectrophotometric determination of microgram  quantities  of protein  without nucleic  acid  interference.  Anal.  Biochem. 22, 195-210.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem.  193, 265-275.

Folin, O. and Ciocalteu, V.  (1927) Tyrosine and tryptophan determination in proteins. J. Biol. Chem. 73, 627-650.

Peterson, G.  L.  (1979) Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal. Biochem.  100, 201-220.

Smith, P. K., Krohn, R. I., Hemianson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fugimoto, E. K., Goeke, N. M., Olsen, B. J. and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem.  150, 76-85.

Wiechelman, K. J., Braun, R. D. and Fitzpatrick, J. D. (1988) Investigation of the bicinchoninic acid protein assay:  identification of the groups responsible for color formation. Anal. Biochem.  175, 231-237.

Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248-254.

Read, S.  M.  and Northcote, D.  H. (1981) Minimization of variation in the response to different proteins of the Coomassie Blue dye-binding assay  for protein. Anal. Biochem. 116, 53-64.

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علامات مبكرة للإيبولا يمكن الخلط بينها وبين الإنفلونزا

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الآخبار التكنلوجية

الليلة.. هلال القمر يلتقي كوكبي الزهرة والمشتري

إيلون ماسك يخسر دعواه القضائية أمام أوبن إيه.آي

مهمة فضائية "أوروبية صينية" لكشف أسرار الرياح الشمسية

انتهاء رحلة "السفينة الموبوءة".. وهذا مصير من بقي على متنها

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مواضيع ذات صلة

Protein (Western) Blotting

Detection, Estimation and Recovery of Proteins in Gels

Cellulose Acetate Electrophoresis

Other Methods of Protein Purification

Chromatographic Methods for Protein Purification

Producing Recombinant Protein

Engineering Proteins for Purification

حوامض أمينية محورة Modified Amino Acids

Amplification methods

Immunoelectrophoresis

Immunoprecipitation

Antibody production