Mrityunjoy Mahato

Hemoglobin−Silver Interaction and Bioconjugate Formation: A Spectroscopic Study

In this article, we report the results of the extent of interaction as well as the formation of a bioconjugate of human hemoglobin (Hb) with silver (Ag). The complexation process and conformational changes are characterized using different spectroscopic and microscopic techniques. The UV-vis study demonstrates the perturbation of the soret/heme band and generates conformational heterogeneity within the heme group in the presence of silver. A fluorescence study suggests that the Tryptophan (Trp) residues of Hb are in a more polar environment after conjugation. Initial fluorescence enhancement with addition of silver is due to metal-enhanced fluorescence. Moreover, the fluorescence quenching after the formation of the Hb-Ag bioconjugate follows the modified Stern-Volmer (S-V) plot. The S-V plot along with the time-resolved fluorescence study indicates the presence of both static and dynamic types of quenching. In addition, the reduction potential values of the entities (Hb-heme, Ag(+), and Trp) indicate the possible electron transfer. The secondary structure calculation from CD and FTIR spectra indicate alpha-helix to beta-sheet conversion, and unfolding of Hb is also responsible for the bioconjugate formation. In addition, FE-SEM, phase contrast inverted microscopy (PCIM) images demonstrate the formation of the silver-protein bioconjugate. The overall data show that there is a change in the secondary as well as the tertiary structure of Hb after conjugation with silver.

Aggregation Behavior of SDS/CTAB Catanionic Surfactant Mixture in Aqueous Solution and at the Air/Water Interface

Herein, we report the aggregation behavior of catanionic mixtures of the anionic surfactant sodium dodecyl sulfate (SDS) and the cationic surfactant cetyltrimethylammonium bromide (CTAB) in solution and at the air/water interface obtained by the Langmuir-Blodgett (LB) technique. We employed Fourier transform infrared spectroscopy, in situ phase-contrast inverted microscopy, scanning electron microscopy, and atomic force microscopy to characterize the systems in solution, at the air/water interface, and in LB films. We found spherical vesicles at the SDS/CTAB ratio of 35/65 in aqueous solution and an ordered aggregated morphology called surface micelles at SDS/CTAB ratios of 35/65 to 65/35 at the air/water interface. Other mixtures (SDS/CTAB = 90/10, 10/90) were found to contain mostly disordered aggregated microstructures. An in situ time-dependent study of surface micelle formation at the air/water interface showed micelle ripening through the fusion of smaller micelles. These micelles were successfully immobilized on a glass substrate by the LB technique. Overall, the study might find application in the fundamental science of the physical chemistry of surfactant systems, as well as in the preparation of drug delivery system.

Study of silver nanoparticle–hemoglobin interaction and composite formation

Nanoscience is now an expanding field of research and finds potential application in biomedical area, but it is limited due to lack of comprehensive knowledge of the interactions operating in nano-bio system. Here, we report the studies on the interaction and formation of nano-bio complex between silver nanoparticle (AgNP) and human blood protein hemoglobin (Hb). We have employed several spectroscopic (absorption, emission, Raman, FTIR, CD, etc.) and electron diffraction techniques (FE-SEM and HR-TEM) to characterize the Hb-AgNP complex system. Our results show the Hb-AgNP interaction is concentration and time dependent. The AgNP particle can attach/come closer to heme, tryptophan, and amide as well aromatic amine residues. As a result, the Hb undergoes conformational change and becomes unfolded through the increment of β-sheet structure. The AgNP-Hb can form charge-transfers (CT) complex where the Hb-heme along with the AgNP involved in the electron transfer mechanism and form Hb-AgNP assembled structure. The electron transfer mechanism has been found to be dependent on the size of silver particle. The overall study is important in understanding the nano-bio system and in predicting the avenues to design and synthesis of novel nano-biocomposite materials in material science and biomedical area.

Effect of salt on the formation of alcohol-dehydrogenease monolayer: a study by the Langmuir-Blodgett technique.

We report here the effect of salt (KCl) on the interfacial surface activity of yeast alcohol dehydogenease (ADH) at air/water interface using the Langmuir-Blodgett technique. Effect of salt content in the water subphase on ADH structure has been studied. The change of area/molecule, compressibility, rigidity, and unfolding of ADH are insignificant up to 10 mM KCl concentration. The significant changes are observed above 0.1 M KCl concentrations. Observations are explained in the context of DLVO theory. FTIR study of amide band together with AFM imaging of ADH monolayer indicate that KCl perturbs the ADH monolayer by the increment of beta-structure resulting into larger unfolding and intermolecular aggregates at high salt concentration.

The formation of pepsin monomolecular layer by the Langmuir-Blodgett film deposition technique.

We report herein the formation of pepsin monomolecular layer by the Langmuir-Blodgett film deposition technique. An effort was made to find an optimal subphase by adjusting the concentration of salt (KCl) and pH by monitoring the growth kinetics of pepsin for the formation of Langmuir monolayer by using as little as possible pepsin molecules to build up ultra thin film and to measure the extent of denaturation. Significant changes of area/molecule, compressibility, rigidity and unfolding of pepsin are observed at optimized subphase than pure water subphase. Observations at optimal subphase are explained in context of the modified DLVO theory and the site dissociation model. FTIR analysis of amide band together with the observed surface morphology of pepsin film in FE-SEM images indicate that at optimal subphase the pepsin molecules modify their structures by incrementing the beta-structure, resulting into larger unfolding and inter-molecular aggregates.

On the origin of iron-oxide nanoparticle formation using phospholipid membrane template

In this report, we have studied the formation of iron-oxide nanoparticle at biologically relevant phospholipids, DPPC Langmuir monolayer at air/water interface. Water subphase contains FeCl(3). Adsorption and agglomeration of Fe(3+) ions at DPPC head group have being monitored by Langmuir and Langmuir Blodgett (LB) technique. Adsorption kinetics (pi-t) as well as the surface pressure area (pi-A) isotherms measurement demonstrate the incorporation of Fe(3+) ion at DPPC monolayer. The amount of incorporation of Fe(3+) to the DPPC monolayer is FeCl(3) concentration and time dependent. This reaction kinetics is well fitted by single exponential association equation. The composite monolayers transferred to different substrates are characterized by UV-vis absorption spectroscopy and electron microscopy (FE-SEM and HR-TEM). Study shows the formation of monodisperse Fe(3)O(4) nanoparticle having size approximately 20 nm coated with DPPC mono or multilayer. The overall study indicates that the formation as well as assembly of iron-oxide nanoparticle in two dimensions is possible using lipid monolayer as a template.

pH induced structural modulation and interfacial activity of hemoglobin at the air/water interface.

In this Article, we report the surface activity of the human globular blood protein, hemoglobin (Hb), at the air/water interface. The Langmuir-Blodgett technique is used for monolayer characterization. The adsorption growth-kinetics study shows that the adsorption process at the air/water interface is involved with two mechanisms: one diffusion with adsorption and the other rearrangement with unfolding. The kinetics is found to be dependent on pH and protein concentration in the subphase. The CD and FTIR studies suggest larger intermolecular aggregate and beta-sheet formation in the film lifted from the air/acidic water subphase. In alkaline pH and in isoelectric pH (6.8), not much variation is observed. The FE-SEM images support this observation. The acidic pH induced such conformational changes, and aggregation is explained with the argument of alpha-helix to beta-sheet conversion as well as the competition between protonation and deprotonation of the aromatic-amino acid residues at the air/water interface.

Fibrillation of Egg White Ovalbumin: A Pathway via Biomineralization

Here we report the fibrillation of egg white ovalbumin (OVA) induced by the biomineralization of two alkali halides (KCl, NaCl) in the Langmuir-Blodgett (LB) film of OVA. The pressure-area isotherm of OVA shows the salt-induced increment of apparent area/monomer of OVA. Fibrillation of OVA in the LB film is monitored by FE-SEM imaging. Formation of fibrillar aggregates is concomitant with an increase of salt concentration. HR-TEM and EDX measurements allowed us to identify nanostructured crystals of salt, which are associated with this fibrillar structure. FTIR spectroscopic study of the amide band in LB films as well as CD spectroscopy in solution qualitatively indicates the increase in β-sheet to α-helix ratio in the presence of salt, indicating unfolding of protein. We suggest that the ion attachment to the peptide chain leads to unfolding and that subsequent recrystallization in the transferred monolayer leads to fibrillation of protein as well as biomineralization of alkali halide salts. This finding demonstrates that the fibrillation of OVA is induced by the biomineralization of alkali halides.

Interaction of glucose with hemoglobin: a study in aqueous solution and at the air–water interface using the Langmuir–Blodgett technique

Here, we report the glycosylation of human adult hemoglobin (Hb) studied in aqueous solution and at the air-water interface by the Langmuir-Blodgett (LB) technique. Pressure-area (π-A) and pressure-time (π-t) measurements show that the concentration of glucose (GLC) and interaction time have an effect on Hb molecular area as well as on surface activity. Solution studies by UV-vis absorption and emission spectroscopy show that the GLC can alter the local conformation of Hb to some extent at the tryptophan and heme residues. CD spectroscopic studies in solution indicate that the α-helix content increases in the presence of GLC at the secondary structure level, which may be the cause of an increased adsorption rate of Hb. Also, secondary structure calculation using FTIR technique in the LB film follows the decrease in α-helix and increase in β-sheet structure as well as the formation of intermolecular aggregates. AFM images of Hb in the LB film indicate the transition from globular to an ellipsoid-like structure of Hb in the presence of GLC. FTIR studies of the LB film support the AFM imaging and the analysis of π-t kinetics. The molecular docking study revealed that Val 1 and Lys 132 are the most favorable docked sites along with some other sites such as Hem 147, Trp 37, Asp 94, Tyr 145, Leu 91, His 143, Glu 43 etc. The overall study may predict the processes of interactions with the increased concentration of GLC on Hb as well as on other long lived proteins.

PROTEIN MONOLAYER FORMATION AT AIR–ELECTROLYTE INTERFACE: A LANGMUIR–BLODGETT STUDY

The interfacial surface activity of a protein, ovalbumin (OVA) at bare air/water interface in presence and also in absence of electrolyte (KCl) in subphase has been investigated. The surface activity was measured as a function of time. It has been found that, the presence of KCl in aqueous subphase enhances the adsorption rate of the protein. The changes of area/molecule, compressibility, rigidity and unfolding of OVA are trivial up to 10 mM KCl concentration. These properties of OVA, above 10 mM KCl concentration are significant and have been explained in the perspective of DLVO theory and many-body ion–protein dispersion potentials. The presence of high concentration of electrolyte increases the β-structure of OVA, resulting into larger unfolding as well as larger intermolecular aggregates. The overall study indicates that KCl perturbs the OVA monolayer.