Q.53 The molar extinction coefficients of Trp and Tyr at 280 nm are 5690 M−1·cm−1 and 1280 M−1·cm−1, respectively. The polypeptide
chain of yeast alcohol dehydrogenase(37 kDa) contains5 Trp and 14 Tyr residues. The absorbance at 280 nm of a 0.32 mg·mL−1 solution
of yeast alcohol dehydrogenase measured in a cuvette of 1 cm pathlength will be __________.
(Assume that the molar extinction coefficient values for Trp and Tyr apply to these amino acids in yeast alcohol dehydrogenase.)
Wavelength: 280 nm |
Absorbance: ≈ 0.40
Introduction
Proteins such as yeast alcohol dehydrogenase absorb ultraviolet light at
280 nm primarily due to the presence of aromatic amino acids,
especially tryptophan (Trp) and tyrosine (Tyr).
This property allows accurate protein quantification using the
Beer–Lambert law.
Beer–Lambert Law
Equation:
A = ε × C × l
- A = absorbance at 280 nm
- ε = molar extinction coefficient (M-1 cm-1)
- C = molar concentration (M)
- l = pathlength (cm)
Given Data
- Protein molecular weight = 37 kDa = 37,000 g·mol-1
- Protein concentration = 0.32 mg·mL-1 = 0.32 g·L-1
- Number of Trp residues = 5
- Number of Tyr residues = 14
- Pathlength (l) = 1 cm
Step-by-Step Calculation
Step 1: Convert Mass Concentration to Molarity
C = 0.32 g·L-1 / 37,000 g·mol-1
= 8.649 × 10-6 M
Step 2: Calculate Molar Extinction Coefficient (ε280)
ε280 = (5 × 5690) + (14 × 1280)
= 28,450 + 17,920
= 46,370 M-1·cm-1
Step 3: Calculate Absorbance at 280 nm
A280 = ε × C × l
= 46,370 × 8.649 × 10-6 × 1
= 0.401 ≈ 0.40
Option Analysis
- 0.37 – 0.43: Correct. The calculated value (0.401) lies within this range.
- 0.37: Too low; results from rounding or partial contribution errors.
- 0.23: Incorrect; often due to ignoring Trp contribution or pathlength.
- None: Invalid, as Beer–Lambert law gives a clear numerical result.
Final Answer
Absorbance at 280 nm (A280) ≈ 0.40
(Correct option range: 0.37 – 0.43)
Applications in Biotechnology
Absorbance at 280 nm is widely used for protein quantification during purification
and enzymatic studies. Unlike dye-based assays (e.g., Bradford),
this method directly measures intrinsic aromatic residues, making it
essential in molecular biology, enzymology, and biochemical engineering workflows.
