Influence of KCl on the interfacial activity and conformation of hemoglobin studied by Langmuir–Blodgett technique

Abstract

We report here the influence of KCl on the interfacial surface activity and conformation of human adult hemoglobin (Hb) using Langmuir and Langmuir-Blodgett (LB) techniques. The studies were done in absence and in presence of KCl salt in the subphase. We have studied the surface pressure-area (π-A) isotherm and surface pressure-time (π-t) kinetics of Hb with the variation of KCl concentrations (C(KCl)). The π-t study shows that the surface activity as well as the magnitude of diffusion and rearrangement of Hb at air/water interface is a function of C(KCl). Conformational study was done by CD spectroscopy and by the FTIR technique. Both the studies show an increasing trend of the α-helix form of Hb in the presence of KCl which may be responsible for the increased surface activity of Hb. The free energy calculations show that the compression of the Hb monolayer is involved in the small free energy change (∼5-25 kcal mol(-1)) of Hb. The changes in area per molecule and free energy, as well as other results, indicate that the influence of KCl on the Hb monolayer is in line with modified DLVO theory of ion-protein interaction. FE-SEM study shows that the LB films in absence of KCl comprise higher aggregates, whereas in the presence of KCl (0.5 M) it comprises lower aggregates, indicating the structural change of Hb. KCl salt here enhances the α-helix from of Hb, promoting the folded conformation by perturbing the water structure. The overall results show that the intermolecular forces and the surface activity as well as the population of the α-helix of Hb can be tuned by KCl salt.

Growth and time dependent alignment of KCl crystals in Hemoglobin LB monolayer

“…1B]) that the film contained some units of globular structures having comparable dimension with the tetrameric Hb along with some higher aggregates [38]. However, there is some structural change of Hb at the molecular level in presence of salt at the subphase [38].…”

“…As stated earlier that we have studied the details of the p-A isotherms of Hb monolayers at the air/water interface with different C KCl at the subphase [38]. Traces (a) and (b) in Fig.…”

“…In our earlier literature, the changes in surface activity and the conformational change of Hb resulting from KCleHb interactions at the molecular level were reported [38]. The ioneprotein (K þ /Cl À -Hb) interaction has been studied in LB system by measuring the pressureearea isotherm, pressure-time growth kinetics and it was correlated with existing DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory [38,40], regarding ioneprotein interaction.…”

“…The ioneprotein (K þ /Cl À -Hb) interaction has been studied in LB system by measuring the pressureearea isotherm, pressure-time growth kinetics and it was correlated with existing DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory [38,40], regarding ioneprotein interaction. The unfolding of Hb in presence of KCl salt was studied in details.…”

“…The unfolding of Hb in presence of KCl salt was studied in details. Furthermore, we involved in studying the conformation of Hb molecules in LB film in presence of KCl salt at the molecular level using high-resolution field emission scanning electron microscopic (FE-SEM) imaging [38].…”