uv absorbance report

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UV AbsorbanceFor Determination of ProteinConcentration

Paula Denice C. BagunuCAS-02-601P


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INTRODUCTION Ultravioletvisible

spectroscopy or ultraviolet-visiblespectrophotometry (UV-Vis or UV/Vis)refers to absorption spectroscopy orreflectance spectroscopy in the ultraviolet-visible spectral region

Protein, including that in tissues and protein

crystals, absorbs ultraviolet light quite strongly.Rather, it is the amino acids that make up theproteins that absorb the UV light. The strongabsorbance of UV light by protein allows forrapid analysis of protein samples, includingprotein crystals, by microscopy and

microspectroscopy
http://en.wikipedia.org/wiki/Absorption_spectroscopyhttp://en.wikipedia.org/wiki/Ultraviolethttp://en.wikipedia.org/wiki/Visible_spectrumhttp://en.wikipedia.org/wiki/Visible_spectrumhttp://en.wikipedia.org/wiki/Ultraviolethttp://en.wikipedia.org/wiki/Absorption_spectroscopy


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Near UV Absorbance(280 nm)

Quantitation of the amount of protein in asolution is possible in a simple

spectrometer. Absorption of radiation in the near UV by

proteins depends on the Tyr and Trpcontent (and to a very small extent on theamount of Phe and disulfide bonds).

Therefore the A280 varies greatly betweendifferent proteins (for a 1 mg/mL solution,from 0

up to 4 for some tyrosine-rich woolproteins, although most values are in therange

0.5-1.5)


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Advantage:The advantages of thismethod are that it is simple, and thesample is recoverable.

Disadvantage: interference fromother chromophores, and the specific

absorption value for a given proteinmust be determined.

-The extinction of nucleic

acid in the 280-nm region may be asmuch as 10 times that of protein attheir same wavelength, and hence, afew percent of nucleic acid cangreatlyinfluence the absor tion.


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Far UV Absorbance

The peptide bond absorbs strongly in the far UVwith a maximum at about 190 nm. This verystrong absorption of proteins at these wavelengthshas been used in protein determination.

Because of the difficulties caused by absorption byoxygen and the low output of conventionalspectrophotometers at this wavelength,measurements are more conveniently made at

205 nm, where the absorbance is about half thatat 190 nm. Most proteins have extinction coefficients at 205

nm for a 1 mg/mL solution of 30-35 and between20 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 inaddition there is little variation in response between

different proteins, permitting near-absolutedetermination of protein.

Disadvantages: necessity for accurate calibrationof the spectrophotometer in the far UV. Manybuffers and other components, such as heme orpyridoxal 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 lampshould be selected for maximum intensity

at the particular wavelength. 7. Cuvets, quartz, for


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METHODS

Estimation of Protein by Near UVAbsorbance (280 nm)

-A reliable spectrophotometer is necessary. The proteinsolution 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 thatis appropriate for the protein which may already be in aparticular 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 thecuvette. If not 1 cm, it must be adjusted by the appropriatefactor. The Beer-Lambert law states that:

A (absorbance) = c l

where = extinction coefficient, c = concentration inmol/L and l = optical path length in cm. Therefore, if e isknown, measurement of A gives the concentration directly, isnormally quoted for a 1-cm path length.

The actual value of UV absorbance for a given protein mustbe determined by some absolute method, e.g., calculatedfrom the amino acid composition, which can be determinedby amino acid analysis . The UV absorbance for a protein isthen 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 Cysresidues in the polypeptide of mass M and 5690, 1280 and120 are the respective extinction coefficients for theseresidues


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Estimation of Protein by Far UVAbsorbance

The protein solution is diluted with a sodiumchloride solution (0.9% w/v) until the extinction at215 nm is


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TheA2o 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 fromthat at 215 nm; the difference multiplied by144 givesthe 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 1UV MEASUREMENTS OF DNA, RNA AND PROTEINS

WAVELENGTH SIGNIFICANCE COMMENTS215-230 nm Minimum absorbance for

nucleic acids

Peptide bonds in proteinsabsorb light

Measurements aregenerally not performed at

this wavelength becausecommonly used buffersand solvents, such as Tris,also absorb at thesewavelengths.

260 nm Nucleic acids havemaximum absorbance

Purines absorbancemaximum is slightly below260; pyrimidines

maximum. is slightlyabove 260. Purines have ahigher molar absorptivitythan pyrimidines.

Therefore, the absorbancemaximum and absorptivityof a segment of DNAdepends on its basecomposition.Proteins have littleabsorbance at thiswavelength.

270 nm Phenol absorbs strongly Phenol may be acontaminant in nucleicacid preparations.

280 nm Aromatic amino acidsabsorb light

Nucleic acids also havesome absorbance at this


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Proteins have two absorbance peaks in the UV region, onebetween 215-230 nm, where peptide bonds absorb, andanother at about 280 nm due to light absorption by

aromatic amino acids (tyrosine, tryptophan andphenylalanine). Certain of the subunits of nucleic acids(purines) have an absorbance maximum slightly below 260nm while others (pyrimidines) have a maximum slightlyabove 260 nm. Therefore, although it is common to say thatthe absorbance peak of nucleic acids is 260 nm, in reality,

the absorbance maxima of different fragments of DNA varysomewhat 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 aprosthetic group (e.g. Fe2+) or an unnatural amino acid. However, theamino acids tryptophan, tyrosine and cysteine absorb light in the UV

wavelength:Tryptophan has a peak of absorption at 280nm in the UV rangeThis is a useful wavelength to quantitate the absorption of tryptophanSince the absorption is proportional to concentration, this is a usefulway to quantitates protein concentration (for proteins containing Trp)


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Important aspects of quantification of proteins using UVabsorbance

If a protein contains Trp, Tyr or Cys residues it will absorb

in the UV. If it does not contain these amino acids, it willnot absorb UV light, and we cannot quantify it using thismethod

Multiple Trp, Tyr or Cys residues will contribute to theExtinction coefficient for the protein. Thus, we need to

know how many of these residues are present in the proteinto know the correct extinction coefficient

Nucleic acids (DNA, RNA) contaminant will also absorb UVlight, as will other proteins with

Trp, Tyr and Cys residues. Thus, the sample must be

PURE to use UV absorption to quantify a protein.


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Amino Acid E280nm (M-1 cm-1)

Trp 5690

Tyr 1280

Cys 120

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

Example:Bovine insulin contains 4 Tyr residues, 6 Cys residues and 0 Trp residues. We

can determine the expected molar extinction coefficient at 280nm, E280nm, bythe following calculation:E280nm = (0)(5690) + (4)(1280) + (6)(120)E280nm = 5840 M-1 cm-1

Thus, a 1.0M solution of pure bovine insulin would give an absorbance of 5,840 at280nm (obviously, it would have to be diluted considerably to be read accurately).A useful expression relating the parameters of E, concentration (C) and A arederived from the Beer-Lambert law (assuming 1cm path length):A/E = CFor example, if a sample of bovine insulin was observed to give an absorbance at280nm of 0.745 we could calculate the concentration to be:0.745/5840 M-1 cm-1 = CC = 1.28 x 10-4M (note: cm-1 drops out with 1cm pathlength)


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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 thatexposures must be long.

2. For damaging UV light

Weak fluorescence means that protein samples must be exposed todamaging UV light for longer periods of time. Absorbance imagingand spectroscopy is completed rapidly so UV exposure is short andlimited.

3. Tryptophan required for fluorescence

The residue tryptophan has the highest quantum yield of the aminoacids that fluoresce. If it is not present in a high enoughconcentration, there is no detectable fluorescence. Withabsorbance, tryptophan is just one amino acid that absorbs.Additionally, the peptide bonds between the amino acids alsoabsorb!


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4. Fluorescence can be quenched

Impurities, other amino acids, even the proteins structure canconspire to quench fluorescence.

5. Contaminants easily distinguished Salt crystals and other contaminants are easily and rapidly

distinguished from protein crystals by UV absorbance imaging andspectroscopy.

6. Determination of protein concentration

UV absorbance can be used to determine the concentration of theprotein in a crystal.

7. Analysis of DNA and RNA

UV absorbance can be used to analyze DNA and RNA samples andcrystals. Neither DNA nor RNA fluoresce.

8. Determining the contamination of DNA and RNA samples The contamination of DNA or RNA crystals by protein can be

rapidly and safely measured in a non-destructive fashion. This isnot possible using protein fluorescence.


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Additional infoAdvantages- rapid and sensitive

- nondestructive- no ammonium interference (used to isolateproteins)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 researchpurposes by monitoring the extraction or separationof proteins


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The Importance of using the Appropriate Microplatefor Absorbance Measurements in the Ultraviolet

Region of the Spectrum

Figure 1. Spectral analysis of several different microplates. The indicatedmicroplates were scanned form 200 nm to 800 nm in 1 nm increments using aPowerWave 200 scanning microplate spectrophotometer. In each case scans were

performed on wells containing 100 l of distilled water.


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Chromophore Example Excitation max, nm Solvent

C=C Ethene __> * 171 15,000 hexane

C C 1-Hexyne __> * 180 10,000 hexane

C=O Ethanaln __> *

__> *290180

1510,000

hexanehexane

N=O Nitromethanen __> *

__> *275200

175,000

ethanolethanol

C-X X=BrX=I

Methyl bromideMethyl Iodide

n __> *n __> *

205255

200360

hexanehexane


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The End

uv absorbance report

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