Question

Write an equation for the absorbance of any protein as a function of protein concentration using the Beer-Lambert law. Assume that the protein is pure, you know the primary sequence, and that there are no other components that absorb light at lambda = 280 nm.

Answer 1

Determination of protein concentration using ov-Dis Spectroscopy : OV-UTS spectroscopic quantitation holds good for pure proteins Absur- Phon Absorpiton spectrum of a protein in the near and far UV regims. 250 200 wavelength (nm) → Absorption of UV radiation is usually represented in terms of Absorbance ¢ % transmitance : Absorbance (A) = log 10 1o = Intensity of Insident [= Intensity of % Transmitance = to X160%. Transmitted light. Absorbance of any analyte depends on the concentration of the analyte and the path length of the solutim (Beer- Lambert law) :* (A=&cl E = Molar absorption coefficient C = Molar concentration of the analyse la path length of the cell containing analyte solution a 9F E and I are known, concentration can directly determined by using Beer- Lambert law. The equation for Absorbance of any protein as a function of protein concentratton is given by A= Absorbance conce of protein. CECA

Aromalie amino acich, tryptophan, tyrosine and Phenylalanine and disulphile linkage constitute the chromophore that absorb in the near UV region Absorption of near UV region by proteins is usually monitored at 280 nm. In question it is given that, there are no other components that absorb light at 1 = 280 nm, so the protein is lack or aromatic residue and disculphide linkage. This type protein do not absorb the near VU radiation : The concentration cat such proteins cante be estimated using far ou radiatim. estimated The concentration can be using wadell's method that relies on absorbance at 2150m ¢ 225 nm . Protein concentration center = 144 44215 – A225] A215 - Absorbane at 215 nm A225= Absm bane at 225 nm.