The interaction between proteins and the dye (Coomassie Brilliant Blue G-250) in the Bradford protein assay is both interesting and fundamental to its functioning.

1. Nature of Interaction:

The Coomassie Brilliant Blue G-250 dye exists in three forms, depending on the pH and environment:

• The cationic (red) form at very low pH
• The neutral (green) form at intermediate pH
• The anionic (blue) form at higher pH

In the acidic solution of the Bradford reagent (usually in the presence of phosphoric acid), the Coomassie dye is primarily in its brownish-green, neutral form. When protein is added, the dye undergoes a transition to the anionic blue form.

2. Mechanism of Interaction:

• Electrostatic Interaction: The protein’s basic amino acids, particularly arginine, histidine, and lysine, carry positive charges under acidic conditions. They play a crucial role in the binding of the dye due to the establishment of ionic interactions with the negatively charged sulfonic acid groups on the dye.
• Van der Waals Forces: Along with electrostatic interactions, van der Waals forces also play a role in the stabilization of the protein-dye complex.

3. Resultant Change:

When the Coomassie dye binds to protein, there is an increase in the number of anionic blue forms of the dye. This shift is responsible for the observed color change. The intensity of the blue color produced is proportional to the amount of protein present, and this color can be quantified using a spectrophotometer, typically at a wavelength of 595 nm.

4. Advantages of this Interaction:

• Immediate Readout: The transition of the dye upon binding to proteins is swift, making it a rapid assay.
• Stability: Once the protein-dye complex is formed, the blue color remains stable for some time, allowing flexibility in reading the absorbance.
• Specificity: The dye primarily reacts with the protein and is less affected by the presence of small molecules, making the assay more specific to proteins.

5. Limitations:

• Variability in Binding: Not all proteins bind the dye with equal affinity. This means that, in principle, different proteins could produce slightly different color intensities even if they are at the same concentration. This is one reason BSA or other protein standards are used—to generate a standard curve against which unknown samples can be compared.
• pH Sensitivity: Since the interaction is highly dependent on the protonation state of amino acid residues, the assay is sensitive to pH changes.

In summary, the success of the Bradford assay is based on the molecular interactions between the Coomassie dye and proteins, leading to a detectable and quantifiable color change. This fundamental interaction has made the assay a mainstay in protein biochemistry labs worldwide.