Biochem Practical 12

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Priciples of Protein Estimation

September 25th, 2012


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When a beam monochromatic light passes through a transparent medium, partof the light is absorbed and the transmitted beam has a lower intensity thanthe intensity of the incident beam.

Solutions are placed in containers (called "cuvettes") whose material is

transparent: quartz is commonly used, but for visible light, one also usesdisposable cuvettes made of polystyrene or polycarbonate.

The TRANSMITTANCE, Tof the solution isdefined as the ratio of theintensity of the transmitted beam, I to that

of the incident beam, Io:T = I/Io

The ABSORBANCE, A of a solution is definedas A = -log10T. Since A is a logarithmicfunction, it is dimensionless.


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The Beer-Lambert law:

The Beer-Lambert law (also known as Beer's law) applies to solutions oflight-absorbing substances.

It states that the absorbance is directly proportional to the path length,

of the sample and its concentration:

A = cl

where is the MOLAR EXTINCTION COEFFICIENTof the solute, c isthe molar concentration, and l is the path length.

The molar extinction coefficient:

Constant for a particular solute. It varies with the wavelength of the

light.

ABSORPTION SPECTRUM (both

visible and UV range)


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The Beer-Lambert law is applicable to the determination of theconcentration of known substances, provided that

(i) The molecular extinction coefficient for the substance is known atthe wavelength at which the measurements are carried out.

(ii) That the path length of the solution is known accurately. Commonly,cuvettes with a path length of 1 cm are used, then, the molarconcentration c is simply:

since A = cl

c = A/

Example:

The molar extinction coefficient of tryptophan at 280 nm is 500

M-1cm-1. A solution of tryptophan has an absorbance at 280nm of 0.225. What is the concentration of tryptophan in thatsolution?

Answer: From the Beer-Lambert law, c = A/ = 0.225/500 = 4.5x 10-4 M.


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However, when you are purifying a protein of your interest from amixture, the value of is unknown.

It is more accurate to determine the absorbance of solutions atseveral known concentrations of a solute, and draw a

STANDARDIZATION CURVE.The linear range of the plot can then be used to determine theconcentration of an unknown solution of that solute.


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Deviations from the Beer-Lambert law:

Beer-Lambert relationship does not necessarily hold under all sets ofexperimental conditions.

Readings at high absorbance values are unreliable as absorbance values tend

to infinity as the transmittance tends to zero. Experimental conditions shouldbe such as to keep absorbance readings below 1.5.

For reliable results, always take readings at the peak values of absorptionbands (the band with highest value of , as it ensures maximum sensitivity.So, in the diagram on right, one would use the wavelength '.

At higher concentrations intermolecular interactions can affect the ease withwhich electrons in multiple bonds respond to radiant energy.

If there are suspended particles in the sample, these will cause lightscattering, thereby reducing the transmitted intensity. Also, stray light infaulty equipment can also affect the readings.


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BIURET ASSAY

It is based on the peptide chain rather than the side groups as reactants.

It thus provides a fairly accurate measurement, with little variation fromprotein to protein.

The mechanism of the Biuret reaction is not well understood.

It appears that cupric ion (Cu2+) reacts with the peptide bonds of proteins,producing cuprous ion (Cu+). The Cu+ ions then form a complex with peptide-bonded nitrogens producing a blue color, with maximum absorbance at 550nm.

This assay is of low sensitivity (range 1 to 6 mg/ml).

Also, low amounts of reducing agents (such as mercaptoethanol anddithiothreitol) and detergents interfere with the assay.

BIURET


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Essentially, it is an enhanced biuret assay involving copper chelation.

The exact mechanism of color formation is poorly understood.

When proteins react with alkaline cupric sulfate in the presence oftartrate, a tetradentate copper complex forms from four peptidebonds and one copper atom ("biuret reaction").

Thereafter, when phosphomolybdic-phosphotungstic acid (Folin-phenol reagent) is added, it gets reduced, producing an intense bluecolor.

It is believed that the color enhancement occurs due to the transferof electron from copper complex to the phosphomolybdic-phosphotungstic acid complex.

The final blue color can be measured at any wavelength between650nm and 750nm.

It is best to measure it at 750nm since few other substances

absorb at that wavelength.


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The presence of tyrosine, tryptophan, cysteine, histidine and asparagine in the

protein further enhances the amount of color produced as they contributeadditional reducing equivalents.

The sensitivity of this method is ~10 g/ml.

In spite of being a relative assay subject to interference from Tris buffer,

EDTA, nonionic and cationic detergents, carbohydrate, lipids and some salts, itis probably the most widely used protein estimation method.

The paper published by Lowry et al in 1952 is the most cited paper inBiological sciences.


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Nowadays BCA reagent is usedfor estimating proteins. It isbasically a modified Lowrymethods based on Cu+ chelation.


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Protein Determination by the Bradford Method:

Preferred assay for estimating proteins as it is simple and rapid.

It is comparatively free from interference by common reagents exceptdetergents.

Coomassie Brilliant Blue G-250, reacts primarily to basic (especiallyarginine) and aromatic amino acids.

The Bradford protein assay is performed in two formats: Standard assayand microassay for use with a microplate reader.


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Bovine serum albumin (Square), -globulin (Circle), and ovalbumin(Triangle) using the microassay.

Bradford method doesnt measure thepresence of peptide bonds but detects

specific amino acids, which binds tothe dye.

The dye does not bind to free arginineor lysine, or to peptides smaller thanabout 3000 Da.

Response of proteins in the Bradford assay


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Relative absorbance of various proteins in Bradford assay

E f ( )


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Estimation of proteins by measuring absorbance (280 nm)

Advantages:

It is fast and convenient.

The assay does not consume the protein.

Disadvantages:

It is relatively less sensitive (0.2 to 2 mg/ml).

There may be considerable error, especially for protein mixtures.

Non-protein components absorbing ultraviolet light intefere with the assay.

Other factors such as pH, ionic strength, etc. can alter the absorbance.

Analysis:

Since A = cl, Concentration = A280nm/Absorbance coefficients of some common protein standards:

Bovine serum albumin (BSA): 63, Bovine, human, or rabbit IgG: 138,Chicken ovalbumin:70.

For protein mixtures in a cell lysate, following formula is used for a roughestimate of protein concentration.

Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260); The second

component is for the absorbance by the nucleic acid


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Absorbance at 205 nm (A205)

Sensitivity is higher at 205 nm compared to 280 nm.

There is little variation between proteins at 205 nm, becausepeptide bonds are measured.

Like A280, A205 is linear with the protein concentration.

The A205 is also rapid and non-destructive.

However, the disadvantage of the A205 is that it isincompatible with most chemicals such as common buffers suchas phosphate and Tris (50 mM) interfere with the A205.

Concentration of the protein is roughly calculated as:

Concentration (mg/ml) = 31 X A205


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I: bovine Immunoglobulin G,B: Bovine serum albumin,

G: Gelatin.


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Sensitivity range:

Biuret assay: 1 to 6 mg/ml.

Absorbance at 280 nm (A280): 0.2 to 2 mg/ml.Lowrys method: ~10g/ml

Bradford protein assay: 10-1000 g/ml (ng)

Fluoroescamine dye assay: pg quantities


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Purification Table for a Horse Radish Peroxidase

STEPS Volume(ml) Total Protein (mg) Activity(units) Specific Activity(units/mg)

Homogenate 1,400 10,000 100,000 10

Precipitate 280 3,000 96,000 32

Ion-exchangechromatography90 400 80,000 200

Gel filtration 80 100 60,000 600

Affinitychromatography6 3 45,000 15,000

Enzyme Unit (international) of Enzyme Activity: Amount of protein which converts 1micromole of substrate to product per min at 250C at optimal pH.- 1 unit Urease (IU) will liberate 1.0 mole of ammonia from

urea per minute at pH 7.0 at 25C.

Unit of Specific Activity: The number micromoles converted per min per mg proteini.e., Units(as above) of enzyme activity per mg protein


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Methods Enzymol. 2009;463:73-95.Quantitation of protein.Noble JE, Bailey MJ.

National Physical Laboratory, Teddington, Middlesex, United Kingdom.

AbstractThe measurement of protein concentration in an aqueous sample is an importantassay in biochemistry research and development labs for applications rangingfrom enzymatic studies to providing data for biopharmaceutical lot release.Spectrophotometric protein quantitation assays are methods that use UV and

visible spectroscopy to rapidly determine the concentration of protein, relativeto a standard, or using an assigned extinction coefficient. Methods aredescribed to provide information on how to analyze protein concentration usingUV protein spectroscopy measurements, traditional dye-based absorbancemeasurements; BCA, Lowry, and Bradford assays and the fluorescent dye-based assays; amine derivatization and detergent partition assays. The

observation that no single assay dominates the market is due to specificlimitations of certain methods that investigators need to consider beforeselecting the most appropriate assay for their sample. Many of the dye-basedassays have unique chemical mechanisms that are prone to interference fromchemicals prevalent in many biological buffer preparations. A discussion ofwhich assays are prone to interference and the selection of alternativemethods is included.

Talanta 2012 Aug 30;98:123 9
http://www.ncbi.nlm.nih.gov/pubmed?term=%22Noble%20JE%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Bailey%20MJ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Bailey%20MJ%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Noble%20JE%22%5BAuthor%5Dhttp://www.ncbi.nlm.nih.gov/pubmed/22939137http://www.ncbi.nlm.nih.gov/pubmed/22939137


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Talanta. 2012 Aug 30;98:123-9.

Systematic comparisons of various spectrophotometric and colorimetric methods tomeasure concentrations of protein, peptide and amino acid: Detectable limits, lineardynamic ranges, interferences, practicality and unit costs.Chutipongtanate S, Watcharatanyatip K, Homvises T, Jaturongkakul K, Thongboonkerd V.

Source

Medical Proteomics Unit, Siriraj Hospital, Mahidol University, Bangkok, Thailand; Center forResearch in Complex Systems Science, Mahidol University, Bangkok, Thailand.

AbstractThere is limited and inconclusive information regarding detectable limits and linear dynamic ranges

of various quantitative protein assays. We thus performed systematic comparisons of seven

commonly used methods, including direct spectrophotometric quantitation at 205 and 280nm

(A205 and A280, respectively), bicinchoninic acid (BCA), Biuret, Bradford, Lowry and Ninhydrin

methods. Bradford method was the most sensitive assay (LOD=0.006mg/ml) and had the widest

range of detectability (LOD-UOD=0.006-100mg/ml) for purified protein and complex protein mixture.

For peptide, A205, A280, Lowry and Ninhydrin methods had a comparable LOD (0.006mg/ml), but

Ninhydrin method had the widest detectability range (LOD-UOD=0.006-100mg/ml). For amino acid,

A205 and Ninhydrin methods had a comparable LOD (0.006mg/ml), but A205 had a wider

detectability range (LOD-UOD=0.006-6.250mg/ml). Biuret method offered the widest linear dynamic

range for purified protein and complex protein mixture (0.391-100mg/ml), A280 offered the widestlinear dynamic range for peptide (0.024-6.250mg/ml), and Ninhydrin method offered the widest

linear dynamic range for amino acid (0.024-0.195mg/ml). Both Laemmli's and 2-D lysis buffers had

dramatic interfering effects on all assays. Concerning the practicality and unit costs, A205 and A280

were the most favorable. Among the colorimetric methods, Bradford method consumed the least

amount of samples and shortest analytical time with the lowest unit cost. These are the most

extensive comparative data of commonly used quantitative protein assays that will be useful for

selecting the most suitable method for each study.
http://www.ncbi.nlm.nih.gov/pubmed/22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Chutipongtanate%20S%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Watcharatanyatip%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Homvises%20T%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Jaturongkakul%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Thongboonkerd%20V%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Thongboonkerd%20V%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Jaturongkakul%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Homvises%20T%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Watcharatanyatip%20K%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed?term=Chutipongtanate%20S%5BAuthor%5D&cauthor=true&cauthor_uid=22939137http://www.ncbi.nlm.nih.gov/pubmed/22939137

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Biochem Practical 12