Tyrosine, Phenylalanine, and Tryptophan are the amino acids that contribute the maximum to spectrophotometric measurement of proteins at 280 nm.

Question Solution

This multiple-choice question tests knowledge of UV absorbance in protein quantification, where aromatic amino acids dominate due to their conjugated ring systems absorbing strongly at 280 nm.

Option Analysis

• Option 1: Tyrosine, Phenylalanine, Tryptophan
  Correct. Tryptophan shows the strongest absorbance (ε ≈ 5500 M⁻¹ cm⁻¹), tyrosine next (ε ≈ 1400 M⁻¹ cm⁻¹), and phenylalanine weakest (ε ≈ 200 M⁻¹ cm⁻¹) among amino acids. All three aromatic residues drive A280 readings, with Trp dominating protein spectra.

• Option 2: Tyrosine, Serine, Threonine
  Incorrect. Serine and threonine lack aromatic rings and have negligible absorbance at 280 nm; only tyrosine contributes significantly here.

• Option 3: Phenylalanine, Alanine, Leucine
  Incorrect. Alanine and leucine are non-aromatic aliphatics with no 280 nm absorbance; phenylalanine contributes minimally compared to Trp/Tyr.

• Option 4: Histidine, Proline, Tryptophan
  Incorrect. Histidine (imidazole) and proline (cyclic) absorb weakly below 280 nm; tryptophan contributes, but the others do not match the trio’s impact.

Proteins are quantified routinely via spectrophotometric measurement at 280 nm (A280), relying on specific amino acids’ UV absorbance from aromatic rings. In spectrophotometric measurement of proteins, tyrosine, phenylalanine, and tryptophan contribute the maximum due to their π-conjugated systems.

Key Absorbing Amino Acids

Tryptophan (Trp) provides the highest molar absorptivity, making it the primary contributor—its indole ring peaks sharply at 280 nm. Tyrosine (Tyr) follows with its phenolic ring, while phenylalanine (Phe) adds lesser but detectable absorbance via its benzene ring. Disulfide bonds and other residues are minor.

Why 280 nm?

This wavelength targets aromatic residues without interference from peptide bonds (≈220 nm). Proteins vary in content (e.g., 1-2% Trp), so extinction coefficients are calculated: A280 = (n_Trp × 5500 + n_Tyr × 1400 + n_Phe × 200)/path length.

Practical Applications

In labs, A280 estimates concentration (e.g., 1 A280 ≈ 1 mg/mL for IgG). For pure proteins, it’s quick and reagent-free; fluorescence enhances sensitivity via Trp/Tyr emission. Exam prep tip: Always prioritize Trp > Tyr > Phe for such questions.