Monday, April 13, 2009

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.
Before measure tryptophan content you have to prior hydrolyze the protein sample, see the method in my previous posting.

Here is the method of the measurement of Tryptophan content:
  1. The protein sample is made 0.1M in NaOH.
  2. 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.
  3. Measure the absorbance at 294.4 and 280 nm in cuvets (transparent to this wavelength, i.e., quartz) in a spectrometer.
  4. 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


    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.
  5. 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: Number 6 in References