The molecular-level insight of protein adsorption and its kinetics at interfaces is crucial because of its multifold role in diverse fundamental biological processes and applications. In the present study, the sum frequency generation (SFG) vibrational spectroscopy has been employed to demonstrate the adsorption process of bovine hemoglobin (BHb) protein molecules at the air–water interface at interfacial isoelectric point of the protein. It has been observed that surface coverage of BHb molecules significantly influences the arrangement of the protein molecules at the interface. The time-dependent SFG studies at two different frequencies in the fingerprint region elucidate the kinetics of protein denaturation process and its influence on the hydrogen-bonding network of interfacial water molecules at the air–water interface. The initial growth kinetics suggests the synchronized behavior of protein adsorption process with the structural changes in the interfacial water molecules. Interestingly, both the events carry similar characteristic time constants. However, the conformational changes in the protein structure due to the denaturation process stay for a long time, whereas the changes in water structure reconcile quickly. It is revealed that the protein denaturation process is followed by the advent of strongly hydrogen-bonded water molecules at the interface. In addition, we have also carried out the surface tension kinetics measurements to complement the findings of our SFG spectroscopic results.
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