UV Absorbance

For Determination of Protein Concentration Click to edit Master subtitle style

Paula Denice C. Bagunu CAS-02-601P 3/4/12

INTRODUCTION

Ultravioletvisible spectroscopyorultraviolet-visible spectrophotometry(UV-VisorUV/Vis) refers toabsorption spectroscopyor reflectance spectroscopy in theultravioletvisiblespectral region Protein, including that in tissues and protein crystals, absorbs ultraviolet light quite strongly. Rather, it is the amino acids that make up the proteins that absorb the UV light. The strong absorbance of UVlight by protein allows for rapid analysis of protein samples, including protein crystals, by microscopy and microspectroscopy

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Near UV Absorbance (280 nm)

Quantitation of the amount of protein in a solution is possible in a simple spectrometer

Absorption of radiation in the near UV by proteins depends on the Tyr and Trp content (and to a very small extent on the amount of Phe and disulfide bonds). Therefore the A280 varies greatly between different proteins (for a 1 mg/mL solution, from 0 up to 4 for some tyrosine-rich wool proteins, although most values are in the range 0.5-1.5 )

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Advantage: The advantages of this method are that it is simple, and the sample is recoverable. Disadvantage: interference from other chromophores, and the specific absorption value for a given protein must be determined. -The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid cangreatly 3/4/12 influence the absorption.

Far UV Absorbance

The peptide bond absorbs strongly in the far UV with a maximum at about 190 nm. This very strong absorption of proteins at these wavelengths has been used in protein determination. Because of the difficulties caused by absorption by oxygen and the low output of conventional spectrophotometers at this wavelength, measurements are more conveniently made at 205 nm, where the absorbance is about half that at 190 nm. Most proteins have extinction coefficients at 205 nm for a 1 mg/mL solution of 30-35 and between 20 and 24 at 210 nm
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Various side chains, including those of Trp, Phe, Tyr, His, Cys, Met, and Arg (in descending order), make contributions to the A205 . Advantages: simplicity and sensitivity. - the sample is recoverable and in addition there is little variation in response between different proteins, permitting near-absolute determination of protein. Disadvantages: necessity for accurate calibration of the spectrophotometer in the far UV. Many buffers and other components, such as heme or pyridoxal groups, absorb strongly in this region.

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Materials

1. 0.1MK2SO 4 (pH 7.0). 2. 5 mM potassium phosphate buffer, pH 7.0. 3. Nonionic detergent (0.01% Brij 35) 4. Guanidinium-HC1. 5. 0.2-1am Millipore (Watford, UK) filter. 6. UV-visible spectrometer: The hydrogen lamp should be selected for maximum intensity at the particular wavelength. 7. Cuvets, quartz, for <215 nm.

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METHODS

Estimation of Protein by Near UV Absorbance (280 nm) - A reliable spectrophotometer is necessary. The protein
solution must be diluted in the buffer to a concentration that is well within the accurate range of the instrument - The protein solution to be measured can be in a wide range of buffers, so it is usually no problem to find one that is appropriate for the protein which may already be in a particular buffer required for a purification step or assay for enzyme activity

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- The value obtained will depend on the path length of the cuvette. If not 1 cm, it must be adjusted by the appropriate factor. The Beer-Lambert law states that: A (absorbance) = c l where = extinction coefficient, c = concentration in mol/L and l = optical path length in cm. Therefore, if e is known, measurement of A gives the concentration directly, is normally quoted for a 1-cm path length. The actual value of UV absorbance for a given protein must be determined by some absolute method, e.g., calculated from the amino acid composition, which can be determined by amino acid analysis . The UV absorbance for a protein is then calculated according to the following formula: A280 (1 mg/mL) = (5690nw + 1280ny + 120nc)/M where n w, ny, and n c are the numbers of Trp, Tyr, and Cys residues in the polypeptide of mass M and 5690, 1280 and 120 are the respective extinction coefficients for these residues

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Estimation of Protein by Far UV Absorbance

The protein solution is diluted with a sodium chloride solution (0.9% w/v) until the extinction at 215 nm is <1.5 Alternatively, dilute the sample in another non-UVabsorbing buffer such as 0.1MK2SO4, containing 5 mM potassium phosphate buffer adjusted to pH 7.0 Measure the absorbances at the appropriate wavelengths (either A280 and A205, or A225 and A215, depending on the formula to be applied), using a spectrometer fitted with a hydrogen lamp that is accurate at these wavelengths, using quartz cuvets filled with a volume of solution 3/4/12 sufficient to cover the aperture through which the

The A2o 5 for a 1 mg/mL solution of protein (,42051 mg/mL)can be calculated within +2%, A2051 mg/mL = 27 + 120 (A280/A205) Alternatively, measurements may be made at longer wavelengths : Protein concentration (g/mL) = 144 (A215 -A225) The extinction at 225 nm is subtracted from that at 215 nm; the difference multiplied by144 gives the protein concentration in the sample in ~g/mL. With a particular protein under specific conditions accurate measurements of concentration to within 5 ~tg/L are possible.

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TABLE 1 UV MEASUREMENTS OF DNA, RNA AND PROTEINS WAVELENGTH SIGNIFICANCE COMMENTS 215-230 nm Minimum absorbance for Measurements are nucleic acids generally not performed at Peptide bonds in proteins this wavelength because absorb light commonly used buffers and solvents, such as Tris, also absorb at these wavelengths. 260 nm Nucleic acids have Purines absorbance maximum absorbance maximum is slightly below 260; pyrimidines maximum. is slightly above 260. Purines have a higher molar absorptivity than pyrimidines. Therefore, the absorbance maximum and absorptivity of a segment of DNA depends on its base composition. Proteins have little absorbance at this wavelength. 270 nm Phenol absorbs strongly Phenol may be a contaminant in nucleic acid preparations. 280 nm Aromatic amino acids Nucleic acids also have 3/4/12 absorb light some absorbance at this

Proteins have two absorbance peaks in the UV region, one between 215-230 nm, where peptide bonds absorb, and another at about 280 nm due to light absorption by aromatic amino acids (tyrosine, tryptophan and phenylalanine). Certain of the subunits of nucleic acids (purines) have an absorbance maximum slightly below 260 nm while others (pyrimidines) have a maximum slightly above 260 nm. Therefore, although it is common to say that the absorbance peak of nucleic acids is 260 nm, in reality, the absorbance maxima of different fragments of DNA vary somewhat depending on their subunit composition. "

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Proteins

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Proteins do not absorb in the visible wavelength unless they have a prosthetic group (e.g. Fe2+) or an unnatural amino acid. However, the amino acids tryptophan, tyrosine and cysteine absorb light in the UV wavelength: Tryptophan has a peak of absorption at 280nm in the UV range This is a useful wavelength to quantitate the absorption of tryptophan Since the absorption is proportional to concentration, this is a useful way to quantitates protein concentration (for proteins containing Trp)

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Important aspects of quantification of proteins using UV absorbance If a protein contains Trp,TyrorCysresidues it will absorb in the UV.If it does not contain these amino acids, it will not absorb UV light, and we cannot quantify it using this method Multiple Trp,TyrorCysresidues will contribute to the Extinction coefficient for the protein.Thus, we need to know how many of these residues are present in the protein to know the correct extinction coefficient Nucleic acids (DNA, RNA) contaminant will also absorb UV light, as will other proteins with Trp,TyrandCysresidues.Thus, the sample must be PURE to use UV absorption to quantify a protein.

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Molar extinction coefficients of Trp, Tyr and Cys amino acids:

Amino Acid Trp Tyr Cys E280nm (M-1 cm-1) 5690 1280 120

Example: Bovine insulin contains 4Tyrresidues, 6Cysresidues and 0 Trp residues.We can determine the expected molar extinction coefficient at 280nm, E280nm, by the following calculation: E280nm= (0)(5690) + (4)(1280) + (6)(120) E280nm= 5840 M-1cm-1 Thus, a 1.0M solution of pure bovine insulin would give an absorbance of 5,840 at 280nm (obviously, it would have to be diluted considerably to be read accurately). A useful expression relating the parameters of E, concentration (C) andAare derived from the Beer-Lambert law (assuming 1cm path length): A/E = C For example, if a sample of bovine insulin was observed to give an absorbance at 280nm of 0.745 we could calculate the concentration to be: 0.745/5840 M-1cm-1= C C = 1.28 x 10-4M (note: cm-1drops out with 1cmpathlength)

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Advantages of UV Absorption of Protein

1. Speed Protein usually absorbs strongly at 280 nm. This means that images and spectra can be acquired quickly. On the other hand, the intrinsic fluorescence of proteins is weak meaning that exposures must be long. 2. For damaging UVlight Weak fluorescence means that protein samples must be exposed to damaging UVlight for longer periods of time. Absorbance imaging and spectroscopy is completed rapidly so UVexposure is short and limited. 3. Tryptophan required for fluorescence The residue tryptophan has the highest quantum yield of the amino acids that fluoresce. If it is not present in a high enough concentration, there is no detectable fluorescence. With absorbance, tryptophan is just one amino acid that absorbs. Additionally, the peptide bonds between the amino acids also absorb! 3/4/12

4. Fluorescence can be quenched Impurities, other amino acids, even the proteins structure can conspire to quench fluorescence. 5. Contaminants easily distinguished Salt crystals and other contaminants are easily and rapidly distinguished from protein crystals by UV absorbance imaging and spectroscopy. 6. Determination of protein concentration UV absorbance can be used to determine the concentration of the protein in a crystal. 7. Analysis of DNA and RNA UV absorbance can be used to analyze DNAand RNAsamples and crystals. Neither DNAnor RNAfluoresce. 8. Determining the contamination of DNA and RNAsamples The contamination of DNAor RNAcrystals by protein can be rapidly and safely measured in a non-destructive fashion. This is not possible using protein fluorescence.

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Additional info Advantages - rapid and sensitive - nondestructive - no ammonium interference (used to isolate proteins) Disadvantages - nucleic and phenolic acids also absorb at 280 nm - amounts of Trp and Tyr vary with protein types - turbidity (cloudiness in solution) is a problem

Applications of method? Not widely accepted for general food analysis more useful for research purposes by monitoring the extraction or separation of proteins
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The Importance of using the Appropriate Microplate for Absorbance Measurements in the Ultraviolet Region of the Spectrum

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Figure 1. Spectral analysis of several different microplates.The indicated microplates were scanned form 200 nm to 800 nm in 1 nm increments using a PowerWave 200 scanning microplate spectrophotometer. In each case scans were performed on wells containing 100 l of distilled water.

Chromophore

Example

Excitation

max, nm

Solvent

C=C CC

Ethene 1-Hexyne

__> * __> *

171 180

15,000 10,000

hexane hexane

C=O

Ethanal

n __>* __>*

290 180

15 10,000

hexane hexane

N=O

Nitromethane

n __>* __>*

275 200

17 5,000

ethanol ethanol

C-X X=Br X=I

Methyl bromide Methyl Iodide

n __>* n __>*

205 255

200 360

hexane hexane

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The End

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