Protein assays — In the Bradford (Coomassie Brilliant Blue G-250) method, the dye binds most strongly to which feature of proteins to produce the characteristic blue shift?

Introduction / Context:
The Bradford assay is a rapid colorimetric method for estimating protein concentration. It relies on the binding of Coomassie Brilliant Blue G-250 to proteins, causing a spectral shift from reddish to blue. Understanding what the dye recognizes helps interpret assay variability across proteins.

Given Data / Assumptions:

• Dye–protein interactions involve electrostatic and hydrophobic forces.
• Proteins differ in amino-acid composition; the assay response can vary accordingly.
• We seek the dominant binding determinant.

Concept / Approach:
Coomassie G-250 preferentially binds to basic and aromatic residues, with the strongest affinity for arginine due to the guanidinium group forming strong electrostatic and stacking interactions with the dye's sulfonated aromatic system. Lesser contributions arise from lysine, histidine, and hydrophobic contacts with aromatic residues, but arginine content best predicts assay response.

Step-by-Step Solution:

Verification / Alternative check:
Comparative calibration using proteins with known arginine content (e.g., BSA vs. IgG) shows differing responses per mass, consistent with arginine-driven binding.

Why Other Options Are Wrong:

• Free C-termini: present once per chain; not the main binding site.
• Peptide bonds: Lowry/Biuret assays target peptide bonds; Bradford relies primarily on side chains.
• Aromatic ring: contributes, but less decisive than arginine's guanidinium interactions.

Common Pitfalls:
Assuming all proteins yield identical absorbance per mass; composition differences, especially arginine content, alter the assay response, necessitating matched standards when possible.

Final Answer:
arginine residues