1. What is Extinction Coefficient?

The extinction coefficient (ε) is a measure of how strongly a chemical species absorbs light at a given wavelength. For proteins, the extinction coefficient at 280 nm is primarily determined by the content of aromatic amino acids: tryptophan, tyrosine, and cysteine.

2. How Does the Calculator Work?

The calculator uses the following formula:

Where:

• \( ε \) — Extinction coefficient (M⁻¹ cm⁻¹)
• \( n_{AA} \) — Number of specific amino acid residues
• \( ε_{AA} \) — Molar extinction coefficient of amino acid

Standard Values at 280 nm:

• Tryptophan (W): 5500 M⁻¹ cm⁻¹
• Tyrosine (Y): 1490 M⁻¹ cm⁻¹
• Cysteine (C): 125 M⁻¹ cm⁻¹ (when in disulfide bonds)

3. Importance of Extinction Coefficient

Details: The extinction coefficient is essential for quantifying protein concentration using UV absorbance at 280 nm. It allows researchers to determine protein concentration without the need for standard curves or colorimetric assays.

4. Using the Calculator

Tips: Enter the protein amino acid sequence using single-letter codes. The sequence should contain only valid amino acid characters (A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y). The calculator will automatically count aromatic residues and compute the extinction coefficient.

5. Frequently Asked Questions (FAQ)

Q1: Why are only W, Y, and C considered?
A: These three amino acids are the primary contributors to UV absorbance at 280 nm due to their aromatic side chains.

Q2: How accurate is this calculation?
A: This provides a theoretical estimate. Actual extinction coefficients can vary due to protein folding, solvent conditions, and local environment effects on aromatic residues.

Q3: What about cysteine contributions?
A: Cysteine only contributes significantly when involved in disulfide bonds. For reduced cysteine, the contribution is negligible.

Q4: Can I use this for protein concentration determination?
A: Yes, using Beer-Lambert law: Concentration = A280 / (ε × path length).

Q5: Are there limitations to this method?
A: This method assumes all aromatic residues are exposed and contributing fully. It may be less accurate for proteins with unusual chromophores or significant light scattering.