Bioinformatics

Biological Basics

1. Amino acids I 2. Peptide bond I 3. Proteins I 4. DNA I 5. Miscellaneous

3. Proteins

• macromolecules / polymers
• 'workhorses of the cell'
• made up of various L-amino acids connected by peptide bonds
• linear chains (unbranched)
  • protein molecule that consists of a single polypeptide chain is said to be monomeric
  • proteins made up of more than one polypeptide chain are called oligomeric
• interact with other proteins, nucleic acids, small molecules, ions, etc.

Protein Quantitation

a.) spectroscopic methods

some aromatic amino acids absorb light in the ultraviolet region proteins have typically UV absorbance maxima around 275-280 nm almost entirely due to the absorbance properties of tryptophan, tyrosine and phenylalanine molar absorptivity of tryptophan is 5 times that of tyrosine, and 50 times that of phenylalanine (log scale ordinate) if you know the molar absorptivity of a particular protein, then you can readily determine the protein concentration by UV-visible spectrophotometry and Beers law (A = εbc) !

ABSORPTION measurement (A280 method)/280 nm

used for protein concentrations of up to 4 mg/ml easy to use and quick method less sensitive then colorimetric methods interference from overlapping absorptions from non-protein substances (e.g. DNA)

Ultraviolet Absorbance (by B. Olson, University of Nebraska-Lincoln)

b.) chemical methods

KJELDAHL method (total nitrogen content)

KJELDAHL method based on sulfuric acid mineralization and measurement of the released ammonia invented in 1883 by a Danish chemist, Johan G.C.T. Kjeldahl analyzing the content of nitrogenous compounds in urine, serum, or other specimens, usually to determine relatively large amounts of nitrogen (e.g., 20 to 100 mg) procedure: treated with a digestion mixture (copper sulfate and sulfuric acid), heated thoroughly, and made alkaline with a solution of sodium hydroxide distilled ammonia from the mixture, trapped it in a boric acid-indicator solution, and titrated with standard hydrochloric or sulfuric acid

A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus (by Labconco Corp.)

c.) colorimetric methods

• protein samples often consist of complex mixtures of different proteins
• quantitative protein content determination of such raw protein solutions is frequently performed by means of reaction between functional groups of the proteins with chromogenic reagents
  • chemistry creates a measurable color in the visible range
• amount of the colored complex formed is proportional to the amount of protein

BIURET method (colorimetric method)/540 nm

oldest method, first described by A.G. Gornall Gornall, A.G.; Bardawill, C.J.; David, M.M.; J. Biol. Chem. 1949, 177(2), 751-766. [PDF] least sensitive (100 to 1000 fold less than LOWRY, BCA or BRADFORD) used for measuring high concentrations of protein in solution (range: 1-5 mg protein/ml) basis for LOWRY and BCA method Cu2+-sulfate in alkaline tartrate reacts with peptide bonds to produce a purple compound (copper-protein complex) with maximum absorption at 540 nm 2 reactions involved: 1. chelation Cu2+/OH- + protein —> [Cu2+-protein complex] 2. redox reaction Cu2+ + (polar amino acids, Trp, Tyr)red —> Cu+ + (amino acids)ox

The Biuret Method (Pierce Chemical Technical Library) Matsushita, M.; Irino, T.; Komoda, T.; Sakagishi, Y.; Clin. Chim. Acta 1993, 216(1-2), 103-111. Determination of proteins by a reverse biuret method combined with the copper-bathocuproine chelate reaction. [PubMed] Sapan, C.V.; Lundblad, R.L.; Price, N.C.; Biotechnol. Appl. Biochem. 1999, 29, 99-108. Colorimetric protein assay techniques. [PDF I PubMed]

BCA method (colorimetric method)

same redox reaction (BIURET method) generates Cu+ BCA forms an intensive purple complex with Cu+ Smith, P.K.; Krohn, R.I.; Hermanson, G.T.; Mallia, A.K.; Gartner, F.H.; Provenzano, M.D.; Fujimoto, E.K.; Goeke, N.M.; Olson, B.J.; Klenk, D.C.; Anal. Biochem. 1985, 150(1), 76-85. Measurement of protein using bicinchoninic acid. [PubMed] BCA = 2,2'-Bicinchoninic Acid or 4,4'-Dicarboxy-2,2'-biquinoline advantages stable color complex applicable over a broad range of protein concentrations less susceptibility to detergents

The BCA Assay (by B. Olson, University of Nebraska-Lincoln) Sapan, C.V.; Lundblad, R.L.; Price, N.C.; Biotechnol. Appl. Biochem. 1999, 29, 99-108. Colorimetric protein assay techniques. [PDF I PubMed] Walker, J.M.; Methods Mol. Biol. 1994, 32, 5-8. The bicinchoninic acid (BCA) assay for protein quantitation. [PubMed]

LOWRY method (colorimetric method)

one of the most common colorimetric assays wide variety of modifications in the literature! first described by O.H. LOWRY Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J.; J. Biol. Chem. 1951, 193, 265-275. Protein measurement with the Folin phenol reagent. much more sensitive than BIURET method protein concentration determination with the Folin-Ciocalteu phenol reagent couples redox reaction (Cu+-formation, BIURET method) with a second redox reaction (color change) LOWRY assay relies on two reactions: formation of a complex between Cu2+ and protein amide (peptide) bonds in an alkaline solution causing a reduction of copper to Cu+ (BIURET reaction) Cu2+ + protein —> [Cu2+-protein complex] Cu2+ + (polar amino acids, Trp, Tyr)red —> Cu+ + (amino acids)ox Cu+ and radical groups of tryptophan, tyrosine, and cysteine reduce a yellow phosphomolybdate-phosphotungstate complex (Folin-Ciocalteu reagent: Na2MoO4 + Na2WoO4 + H3PO4) to a deep blue color Cu+ + (F-C)ox —> Cu2+ + (F-C)red              (F-C) = phospho-Mo-Tungstate acid advantages sensitivity: requiring only 10 to 200 µg of protein number of wavelengths possible for analysis: 500 - 750 nm very good for proteins containing chromophores (hemes, flavins ...) inexpensive reagents critical remarks sensitive to "interference" by many other compounds (contaminants) [interference = production of color by substances other that the analyte of interest] => common problem with indirect colorimetric assays non-linear concentration dependence variations in protein amino acid composition can influence the color development in LOWRY [standard curve by using a protein similar to that being analyzed is required to obtain accurate results] variations in reagent preparation, temperature, and timing require a standard protein curve [must generate each time if the assay is used] LOWRY method is accurate only within ~10 to 200 µg [unknowns with high concentrations of protein must be diluted to fall within this range]

The Lowry Method (Pierce Chemical Technical Library) The Lowry Assay (by B. Olson, University of Nebraska-Lincoln) Alam, A.; Anal. Biochem. 1992, 203(1), 121-126. A model for formulation of protein assay. [PubMed] Sapan, C.V.; Lundblad, R.L.; Price, N.C.; Biotechnol. Appl. Biochem. 1999, 29, 99-108. Colorimetric protein assay techniques. [PDF I PubMed] Waterborg, J.H.; Matthews, H.R.; Methods Mol. Biol. 1994, 32, 1-4. The Lowry method for protein quantitation. [PubMed] Yeang, H.Y.; Yusof, F.; Abdullah, L.; Anal. Biochem. 1998, 265(2), 381-384. Protein purification for the Lowry assay: acid precipitation of proteins in the presence of sodium dodecyl sulfate and other biological detergents. [PubMed]

BRADFORD method (colorimetric method)

BRADFORD reagent consists of Coomassie Brilliant Blue G-250 and phosphoric acid dissolved in ethanol/distilled water according to the method developed by M. Bradford Bradford, M.M.; Anal. Biochem. 1976, 72, 248-254. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. [PubMed] method based on the spectral shift of Coomassie Brilliant Blue maximum absorbance wavelength shifts from 465 nm to 595 nm, when dye is bound to the protein protein stabilizes the anionic form of the dye by hydrophobic and ionic interactions (interaction principally with arginine residues, and to a lesser extent histidine, lysine, tyrosine, tryptophan and phenlyalanine residues) BRADFORD reagent = Coomassie Brilliant Blue G-250 advantages highly sensitive (detect protein in the range from 1-20 ug/ml) simplest to handle and rapid to perform compatibility with reducing agents, which interfere the LOWRY and BCA assay critical remarks significant protein-to-protein variation in absorbance values obtained by the BRADFORD procedure choose a protein standard that is likely to give absorbance values close to those for the protein of interest

Atherton, B.A.; Cunningham, E.L.; Splittgerber, A.G.; Anal. Biochem. 1996, 233(2), 160-168. A mathematical model for the description of the Coomassie brilliant blue protein assay. [PubMed] Bradford, M.M.; Anal. Biochem. 1976, 72, 248-254. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. [PubMed] Chial, H.J.; Splittgerber, A.G.; Anal. Biochem. 1993, 213(2), 362-369. A comparison of the binding of Coomassie brilliant blue to proteins at low and neutral pH. [PubMed] Congdon, R.W.; Muth, G.W.; Splittgerber, A.G.; Anal. Biochem. 1993, 213(2), 407-413. The binding interaction of Coomassie blue with proteins. [PubMed] Friedenauer, S.; Berlet, H.H.; Anal. Biochem. 1989, 178(2), 263-268. Sensitivity and variability of the Bradford protein assay in the presence of detergents. [PubMed] Kruger, N.J.; Methods Mol. Biol. 1994, 32, 9-15. The Bradford method for protein quantitation. [PubMed] Sapan, C.V.; Lundblad, R.L.; Price, N.C.; Biotechnol. Appl. Biochem. 1999, 29, 99-108. Colorimetric protein assay techniques. [PDF I PubMed] Splittgerber, A.G.; Sohl, J.; Anal. Biochem. 1989, 179(1), 198-201. Nonlinearity in protein assays by the Coomassie blue dye-binding method. [PubMed]

Helpful tools

• Protein Composition (by Colorado State University)
  • estimation of the amino acid composition for a given protein sequence/ input

Additional information

• LIFE - The Science of Biology (7th edition) (by W.K. Purves, D. Sadava, G.H. Orians, H. Craig Heller)
• A comparison of reagents for detecting and quantitating proteins in solution. (Molecular Probes, Inc.)
• Brooks, S.P.; Lampi, B.J.; Sarwar, G.; Botting, H.G.; Anal. Biochem. 1995, 226(1), 26-30. A comparison of methods for determining total body protein. [PubMed]
• Cozzone, A.J.; Encyclopedia of Life Sciences 2002, 1-10. Proteins: fundamental chemical properties. [PDF]
• Fountoulakis, M.; Juranville, J.F.; Manneberg, M.; J. Biochem. Biophys. Methods 1992, 24(3-4), 265-274. Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins. [PubMed]
• Jones, L.J.; Haugland, R.P.; Singer, V.L.; Biotechniques 2003, 34(4), 850-854, 856, 858. Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution. [PubMed]
• Knight, M.I.; Chambers, P.J.; Mol. Biotechnol. 2003, 23(1), 19-28. Problems associated with determining protein concentration: a comparison of techniques for protein estimations. [PubMed]

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