28 Feb 2012

The Measurement of Tryptophan Content by Using UV-Spectrometer




The absorption of protein solutions in the UV is the result of tryptophan and tyrosine (and to a very minor, and negligible, extent phenylalanine and cysteine). The absorption maximum will depend on the pH of the solution, and spectrophotometric measurements are usually made in alkaline solutions. Absorption curves for tryptophan and tyrosine show that at the points of intersection, 257 and 294 nm, the extinction values are proportional to the total tryptophan + tyrosine content. Measurements are normally made at 294.4 nm, since this is close to the maximum in the tyrosine curve, and in conjunction with the extinction at 280 nm, the concentrations of each of the two amino acids may be calculated. This is the method of Goodwin and Morton.
Earlier portion tryptophan content you have to prior hydrolyze the protein sample, see the method in my previous posting.

Now is the method of the measurement of Tryptophan content:

The protein sample is made 0.1M in NaOH.

    Absorption by most proteins in 0.1M NaOH solution decreases at longer wavelengths into the region 330--450 nm, where tyrosine and tryptophan do not absorb. Suitable blanks for 294 and 280 nm are therefore obtained by measuring extinctions at 320 and 360 nm and extrapolating back to 294 and 280 nm.
Measure the absorbance at 294.4 and 280 nm in cuvets (transparent to this wavelength, i.E., quartz) in a spectrometer.

The amount of tryptophan (w) is estimated from the relative absorbances at these wavelengths
By the method of Goodwin and Morton (2) shown in Equation below:

    E280 = w Ew + (x-w)Ey

    Therefore:

    W = (E280 - x Ey) / (Ew -Ey)

Where x = total mol/L, w = tryptophan mol/L, and (x- w) = tyrosine mol/L, Ey = Molar extinction of tyrosine in 0.1M alkali at 280 nm= 1576. Ew = Molar extinction of tryptophan in 0.1M alkali at 280 nm = 5225.

Also, x is measured from E294.4 (the molar extinction at this wavelength). This is 2375 for both Tyr and Trp (since their absorption curves intersect at this wavelength). An accurate reading of absorbance at one other wavelength is then sufficient to determine the relative amounts of these amino acids.
An alternative method of obtaining the ratios of Tyr and Trp is to use the formulae derived by Beaven and Holiday.

    MTyr = (0.592 K294- 0.263 K280) / 1000

    MTrp = (0.263 K280 - 0.170 K294) / 1000

Where MTy r and MTrp are the moles of tyrosine and tryptophan in 1 g of protein, and K294 and K280 are the extinction coefficients of the protein in 0.1M alkali at 294 and 280 nm. Extinction values can be substituted for the K values to give the molar ratio of tyrosine to tryptophan according to the formula below:

    MTyr / MTrp = (0.592 E294 - 0.263 E280 / 0.263 E280 - 0.170 E294)


In this analysis, the tyrosine estimate may be high and that of tryptophan low. If amino acid analysis indicates absence of tyrosine, tryptophan is more accurately determined at its maximum, 280.5 nm.


Reference:
    Prieto, L.M. et al., “The production of rhamnolipid by a Pseudomonas aeruginosa strain isolated from a southern coastal zone in Brazil”, Chemosphere (2008), doi:10.1016/j.chemosphere.2008.01.003.
    Burden, D.W.; Whitney, D.B., "Biotechnology, Proteins to PCR, A Course in Strategies and Lab techniques", Birkhauser Boston, 1995.
    Thaniyavarn, J. et al., "Production and Characterization of Biosurfactants from Bacillus licheniformis F2.2" , Biosci. Biotechnol. Biochem., 67 (6), 1239-1244, 2003
    Eeles, R.A.; Stamps, A.C., "Polymerase Chain Reaction (PCR) The Technique And Its Applications", The Institute of Cancer Research, The Royal Marsden Hospital, Sutton, Surrey, United Kingdom, 1993.
    Aitken, A.; Learmonth, M., "The Protein Protocols Handbook, Second Edition: Protein Determination by UV Absorption", Humana Press, 2002.
    Aitken, A.; Learmonth, M., "The Protein Protocols Handbook, Second Edition: Quantitation of Tryptophan in Proteins ", Humana Press, 2002.
    Heptinstall. J and Rapley. R, “The Nucleic Acid Protocols Handbook: Spectrophotometric Analysis of Nucleic Acids”, Humana Press, 2000.
    Walker, J.M., "The Protein Protocols Handbook, Second Edition: The Bicinchoninic Acid (BCA) Assay for Protein Quantitation", Humana Press, 2002.
    Waterborg, J.H., "The Protein Protocols Handbook, Second Edition: The Lowry Method for Protein Quantitation", Humana Press, 2002.
    Kruger, N.J., "The Protein Protocols Handbook, Second Edition: The Bradford Method for Protein Quantitation", Humana Press, 2002.
    Kolmodin, L. A, Williams, J.F, “The Nucleic Acid Protocols Handbook: Polymerase Chain Reaction, Basic Principles and Routine Practice”, Humana Press Inc., Totowa, NJ, 2000.
    Chachaty. E, Saulnier. P, “The Nucleic Acid Protocol Handbook: Bacterial DNA Extraction for Polymerase Chain Reaction and Pulsed-Field Gel Electrophoresis”, Humana Press Inc., Totowa, NJ, 2000.
    Williams. D. R, Rapley. R, “The Nucleic Acid Protocol Handbook: Agarose Gel Electrophoresis of Nucleic Acids”, Humana Press Inc., Totowa, NJ, 2000.
    Smith. D. R, “The Nucleic Acid Protocol Handbook: Restriction Endonuclease Digestion of DNA”, Humana Press Inc., Totowa, NJ, 2000.
    Stewart, T.L., Mann, V., “Methods in Molecular Medicine, Vol. 80: Bone Research Protocols”, Humana Press Inc., Totowa, NJ, 2003.
    Theophilus, B. D. M., “Methods in Molecular Biology, Vol. 86: RNA Isolation and Characterization Protocols”, Humana Press Inc., Totowa, NJ, 1998.
    Perry, J., Parniske, M., "Lotus japonicus Handbook: 96-WELL DNA ISOLATION METHOD", 2005 Springer.
    Winstanley, C., Rapley, R., “The Nucleic Acid Protocols Handbook: Extraction and Purification of Plasmid DNA”, Humana Press Inc., Totowa, NJ, 2000.
    Bogner, P., Killeen, A. A., “Molecular Diagnostics: For the Clinical Laboratorian, Second Edition: Extraction of Nucleic Acids”, Humana Press Inc., Totowa, NJ, 2005.
    Harwood. A. J, “The Nucleic Acid Protocol Handbook: Native Polyacrylamide Gel Electrophoresis”, Humana Press Inc., Totowa, NJ, 2000.
    Aitken. A, Learmonth. M, “The Protein Protocols Handbook, Second Edition: Estimation of Disulfide Bonds Using EIIman's Reagent”, Humana Press Inc., Totowa, NJ, 2002.
    Miller. R. M, Zhang. Y, “Methods in Biotechnology, Vol 2 Bioremediation Protocols: Measurement of Biosurfactant-Enhanced Solubilization and Biodegradation of Hydrocarbons”, Humana Press Inc., Totowa, NJ, 1997.
    Boerner. S. A, Lee. Y. K, Kaufmann. S. H, Bible. K. C., “The Protein Protocols Handbook, Second Edition: The Nitric Acid Method for Protein Estimation in Biological Samples", Humana Press, 2002.




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