How does protein denaturation impact the accuracy of protein quantification methods like the Folin phenol reagent?

Denaturation unfolds the protein, exposing hydrophobic residues and altering how the Folin phenol reagent interacts with the amino acids. The reagent relies on specific interactions with aromatic and sulfhydryl groups; when the protein is unfolded, some groups become hidden or sterically hindered, reducing the signal. Because the reagent’s color intensity is linked to these interactions, a denatured sample often gives a lower absorbance than the same amount of native protein, leading to underestimation. For example, heating a BSA solution before the Lowry assay can result in a 20 % lower reading compared to a native sample. Thus, denaturation can skew quantification unless the method is calibrated for the protein’s state.