A protein has one tryptophan and one, tyrosine in its sequence. Assume molar extinction coefficients at 280 nm of tryptophan and tyrosine as 3000 and 1500 M^-1cm^-l, respectively. What would be the molar concentration of that protein if its absorption at 280 nm is 0.90?
(1) 2 mM                                                       (2) 0.4 mM
(3) 0.2 mM                                                    (4) 0.02 mM

Correct Answer:

(3) 0.2 mM

Topic Explanation: Calculating Protein Concentration Using UV Absorbance and Molar Extinction Coefficients

The concentration of a protein in solution can be calculated from its absorbance at 280 nm using the Beer-Lambert Law:

A=ε×c×l

$$A=\epsilon \times c\times l$$

Where:

• A = absorbance (unitless) at 280 nm

• ε = molar extinction coefficient (M⁻¹cm⁻¹) at 280 nm

• c = molar concentration of the protein (M)

• l = path length of the cuvette (cm), usually 1 cm

Step 1: Calculate total molar extinction coefficient εtotalεtotal:
Given:

• Tryptophan (nW=1 ) contributes 3000 M⁻¹cm⁻¹

• Tyrosine (nY=1) contributes 1500 M⁻¹cm⁻¹

       εtotal      =(1×3000)+(1×1500)=4500 M−1cm−1

Step 2: Rearrange Beer-Lambert law to find concentration $c$:

c=A / ε×l

Assuming pathlength $l$, and A=0.90

c=0.90/ 4500×1

=2×10−4 M

=0.0002 M

=0.2 mM

Summary Table

Why Understanding This Is Important for CSIR NET, ICMR, DBT, IIT JAM and MSc Entrances

This question tests your ability to apply the Beer-Lambert law to calculate protein concentration using intrinsic properties of amino acids, a common task in protein biochemistry. Accurate understanding of molar extinction coefficients and absorbance measurements is foundational for spectroscopic methods widely encountered in:

• CSIR NET Life Sciences

• ICMR and DBT eligibility tests

• IIT JAM Biological Sciences

• MSc entrance exams in Life Sciences

Mastery of these concepts, combined with problem-solving skills, is crucial for acquiring top ranks in competitive life sciences exams.

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In brief:
The molar concentration of the protein is 0.2 mM, making option (3) the correct choice.