Change of the isoelectric point of hemoglobin at the air/water interface probed by the orientational flip-flop of water molecules

Abstract

Elucidation of the molecular mechanisms of protein adsorption is of essential importance for further development of biotechnology. Here, we use interface-selective nonlinear vibrational spectroscopy to investigate protein charge at the air/water interface by probing the orientation of interfacial water molecules. We measured the Im χ spectra of hemoglobin, myoglobin, serum albumin and lysozyme at the air/water interface in the CH and OH stretching regions using heterodyne-detected vibrational sum frequency generation (HD-VSFG) spectroscopy, and we deduced the isoelectric point of the protein by monitoring the orientational flip-flop of water molecules at the interface. Strikingly, our measurements indicate that the isoelectric point of hemoglobin is significantly lowered (by about one pH unit) at the air/water interface compared to that in the bulk. This can be predominantly attributed to the modifications of the protein structure at the air/water interface. Our results also suggest that a similar mechanism accounts for the modification of myoglobin charge at the air/water interface. This effect has not been reported for other model proteins at interfaces probed by conventional VSFG techniques, and it emphasizes the importance of the structural modifications of proteins at the interface, which can drastically affect their charge profiles in a protein-specific manner. The direct experimental approach using HD-VSFG can unveil the changes of the isoelectric point of adsorbed proteins at various interfaces, which is of major relevance to many biological applications and sheds new light on the effect of interfaces on protein charge.

Vibrational spectroscopic study of pH dependent solvation at a Ge(100)-water interface during an electrode potential triggered surface termination transition

“…The existence of "free water" was detected previously by SFG at interfaces between water and hydrophobic liquids, 33,41,[79][80][81][82] or during protein adsorption. 83,84 The observed changes with potential in Fig. 6 are not fully reversible after reverting the potential stepping direction.…”

Förster Resonance Energy Transfer-Mediated Globular Protein Sensing Using Polyelectrolyte Complex Nanoparticles

“…The quenching, i.e., F 0 / F of PEC NPs vs the concentration of all four proteins is shown in Figure 7e, which shows that among the four globular proteins, in the presence of HSA, quenching is higher compared to the others, probably as the isoelectric point ( p I ) of HSA ( p I ≈ 4.7) is lower compared to that of BSA ( p I ≈ 4.9) and Hbg ( p I ≈ 6.8). 39,40 Therefore, the net charge of HSA is more negative compared to the other proteins and exhibits more electrostatic attraction between the positively charged PEC NPs and negatively charged HSA. Although protein takes net positive or negative charges below or above the p I , respectively, both local positive and negative surface charge domains are always present, the relative amount of which depends upon the surrounding pH.…”

Probing the Bovine Hemoglobin Adsorption Process and its Influence on Interfacial Water Structure at the Air–Water Interface

“…14,15 The air–water interface is considered the most hydrophobic in nature, and therefore, it tends to majorly attract the hydrophobic moieties of amphiphilic protein molecules towards the interface. 16−19 Thus, in situ real-time monitoring of the molecular conformation and kinetics of the protein molecules during the air–water interfacial adsorption process is of high importance for many transdisciplinary areas of fundamental research.…”

“…Overcoming the limitations of the conventional techniques, sum frequency generation (SFG) vibrational spectroscopy has been accepted as a powerful technique to investigate the interfacial structure of molecules with excellent molecular and surface specificity at various interfaces. 17,19,32−37 This is a label-free technique that is primarily based on the second-order nonlinear optical process. It uses two pump laser beams in a collinear geometry, i.e., visible (ω Vis ) and tunable IR (ω IR ), which are overlapped in space and time at the interface to produce SFG signal (ω SFG = ω Vis + ω IR ) in the phase matching direction.…”

“…Many fundamental studies related to the molecular structure of a protein and its behavior have been evaluated at the aqueous interface using SFG spectroscopy. 17,19,38−41 Wang et al. used SFG spectroscopy to investigate the conformational changes of bovine serum albumin (BSA) molecules by varying their surface coverage at the air–water interface.…”