Md. Fazle Alam

Study on immobilization of yeast alcohol dehydrogenase on nanocrystalline Ni-Co ferrites as magnetic support.

The covalent binding of yeast alcohol dehydrogenase (YADH) enzyme complex in a series of magnetic crystalline Ni-Co nanoferrites, synthesized via sol-gel auto combustion technique was investigated. The structural analysis, morphology and magnetic properties of Ni-Co nanoferrites were determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), vibrating-sample magnetometer (VSM), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). The comparative analysis of the HRTEM micrographs of bare magnetic nanoferrite particles and particles immobilized with enzyme revealed an uniform distribution of the particles in both the cases without undergoing change in the size which was found to be in the range 20-30 nm. The binding of YADH to Ni-Co nanoferrites and the possible binding mechanism have been suggested by comparing the FTIR results. The binding properties of the immobilized YADH enzyme were also studied by kinetic parameters, optimum operational pH, temperature, thermal stability and reusability. The immobilized YADH exhibits enhanced thermal stability as compared to the free enzyme over a wide range of temperature and pH, and showed good durability after recovery by magnetic separation for repeated use.

Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 μg/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ≥ 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer development.

Molecular hybridization approach of bio-potent CuII/ZnII complexes derived from N, O donor bidentate imine scaffolds: Synthesis, spectral, human serum albumin binding, antioxidant and antibacterial studies

Novel bio-relevant monometallic Schiff base complexes of the type, [Cu(L1)2] (1), [Zn(L1)2]·2H2O (2), [Cu(L2)2]·2H2O (3) and [Zn(L2)2]·H2O (4) [L1(E)-3-(((3-chloro-4-hydroxyphenyl)imino)methyl)naphthalen-2-ol and L2(E)-2-chloro-4-((1-(5-chloro-2-hydroxyphenyl)ethylidene)amino)phenol] were synthesized and characterized. A comparative account of analytical, spectroscopic (FT-IR, 1H and 13C NMR, Mass, UV-vis and EPR), thermal (TGA/DTA), XRD and SEM studies revealed a correlation between the structure and function of these biologically active molecular entities. HSA (Human serum albumin) binding profiles of the metal complexes (1-4) were monitored using biophysical techniques viz., absorbance, fluorescence, circular dichromism (CD) and foster resonance energy transfer (FRET). The intrinsic binding constant (Kb) demonstrated substantial binding propensity of L1 linked complexes (1 and 2) in comparison to L2 complexes (3 and 4) suggesting L1 to be more bio-active pharmacophore due to higher planarity and conjugation as compared to L2 ligand. The outcome of fluorescence study revealed static quenching mechanism on the basis of the quenching of HSA by the complexes (1-4). However, modifications in the secondary structure of HSA by complexes (1-4) inferred via CD measurements which revealed the enhancement of α-helicity (67.47% to 69.20%) with the preference order of 1>2>3>4. Furthermore, in-vitro antibacterial study against different bacteria and antioxidant activities against DPPH and superoxide radical (O2-) at variable concentrations outspread discernible bio-potencies of the metal complexes as compared to free ligand scaffolds due to the chelation effect.

Colorimetric method for the detection of melamine using in-situ formed silver nanoparticles via tannic acid

Melamine toxicity has recently attracted worldwide attention as it causes renal failure and the death of humans and animals. Therefore, developing a simple, fast and sensitive method for the routine detection of melamine is the need of the hour. Herein, we have developed a selective colorimetric method for the detection of melamine in milk samples based upon in-situ formation of silver nanoparticles (AgNPs) via tannic acid. The AgNPs thus formed were characterized by UV-Visible spectrophotometer, transmission electron microscope (TEM), zetasizer and dynamic light scattering (DLS). The AgNPs were used to detect melamine under in vitro condition and in raw milk spiked with melamine. Under optimal conditions, melamine could be selectively detected in vitro within the concentration range of 0.05-1.4μM with a limit of detection (LOD) of 0.01μM, which is lower than the strictest melamine safety requirement of 1ppm. In spiked raw milk, the recovery percentage range was 99.5-106.5% for liquid milk and 98.5-105.5% for powdered milk. The present method shows extreme selectivity with no significant interference with other substances like urea, glucose, glycine, ascorbic acid etc. This assay method does not utilize organic cosolvents, enzymatic reactions, light sensitive dye molecules and sophisticated instrumentation, thereby overcoming some of the limitations of the other conventional methods.

Design, facile synthesis, molecular docking, DNA binding, and cytotoxic activity of polythiophene and polythiophene-titanium(IV) phosphate nanocomposite

ABSTRACT The present article reports the synthesis of polythiophene-titanium(IV) phosphate nanocomposite (PTh-TiP) by a simple in situ titanium(IV) phosphate assisted chemical polymerization of thiophene. The structure and formation of polymer and its nanocomposite was confirmed by FTIR, XRD, FE-SEM, TEM, and TGA techniques. UV-Visible, fluorescence, and circular dichroism spectrophotometry as well as viscometry and DNA melting studies were used to investigate the DNA binding of PTh and PTh-TiP with calf-thymus DNA. The anticancer activities of these materials against MCF-7 and MDA-MB- 231 cancer cells indicated both PTh and PTh-TiP are moderately active, with the latter being more active than the former. GRAPHICAL ABSTRACT

In vitro activity and stability of pure human salivary aldehyde dehydrogenase

Human salivary aldehyde dehydrogenase (HsALDH) appears to be the first line of defence against toxic aldehydes contained in exogenous sources and is important for maintaining healthy oral cavity and protection from oral cancer. Here, the activity and stability of purified hsALDH has been determined under different conditions such as temperature, in presence of denaturants [Urea and guanidine hydrochloride (GnHCl)] and in the presence of salt (NaCl). The pure enzyme exhibited low stability when stored at room temperature (25°C) as well as at low temperature (4°C). 10% glycerol significantly improved its storage stability, particularly at 25°C. HsALDH was observed to have very low thermal stability. At higher temperatures, the enzyme gets unfolded and loses its activity quite rapidly. Further, the enzyme is unstable in the presence of denaturants like urea and GnHCl which unfold the enzyme. Salt (NaCl) has an activating effect on the enzyme, resulting from perhaps due to some conformational changes in the enzyme which facilitates the catalysis process. HsALDH proved to be a labile enzyme under in vitro conditions and certain additives like glycerol and NaCl can improve the activity/stability of the enzyme. Hence, a stabilizing agent is required to use the enzyme in in vitro studies.

In vitro DNA binding studies of therapeutic and prophylactic drug citral

The study of drug-DNA interactions is of great importance, as it paves the way towards the design of better therapeutic agents. Here, the interaction of DNA with a therapeutic and prophylactic drug citral has been studied. We have attempted to ascertain the mode of binding of citral with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of the UV-visible absorbance spectra and fluorescence spectra indicated the formation of a complex between citral and Ct-DNA. Competitive binding assays with ethidium bromide (EB), acridine orange (AO) and Hoechst 33258 reflected that citral possibly intercalates within the Ct-DNA. These observations were further confirmed by circular dichroism (CD) spectral analysis, viscosity measurements, DNA melting and molecular docking studies. This study is expected to contribute to a better understanding of molecular mechanisms of citral, and design of new drugs in the future.

Hybrid pharmacophore approach for bio-relevant di-imines based homobimetallic complexes incorporating functionalized dicarboxylates as co-ligands: Synthesis, spectral and structural activity dependent biological insights (in-vitro DNA and HSA binding, antioxidant and cytotoxicity)

Synthesis of bio-efficient homobimetallic complexes, [Cu2(L1)2(dipic)](NO3)2.3H2O (1), [Zn2(L1)2(dipic)](NO3)2.4H2O (2), [Cu2(L2)2(oxa)](NO3)2.4H2O (3) and [Zn2(L2)2(oxa)] (NO3)2.5H2O (4) was carried out using Schiff bases [(N1E,N2E)-N1,N2-bis(5-chlorothiophen-2-ylmethylene)-4-chlorobenzene-1,2-diamine; L1] and [(N1E,N2E)-N1,N2-bis(5-chlorofuran-2-ylmethylene)-4-chlorobenzene-1,2-diamine; L2] as main ligands and dicarboxylate moieties of 2,6-pyridine dicarboxylic acid (H2-dipic) and oxalic acid (H2-oxa) as co-ligands, respectively in order to apprehend their structure activity relationships on the basis of pharmacophore hybrid approach. The stoichiometry, geometry, thermal stability, morphology and crystallite size of the compounds were inferred by analytical, spectral (FT-IR, 1H NMR and 13C NMR and Mass), thermal (TGA/DTA), SEM and XRD studies. In-vitro DNA and HSA binding profiles of complexes were analysed by different biophysical measurements. The absorption study divulged that the observed alterations in the physico-chemical properties of complexes upon binding with DNA connoted their intercalative binding mode while fluorescence quenching mechanism was quantified by using Stern Volmer constant (KSV); 1.73×104 (1), 1.47×104 (2), 5.65×103 (3) and 3.60×103M-1 (4) which discerned that hybrid pharmacophore active metal complexes (1 and 2) exhibited efficient quenching effect with Ct-DNA in comparison to complexes (3 and 4) due to greater planarity and extent of conjugation (π-π interactions). The intercalative binding mode of complexes is further supported by competitive displacement assay by using fluorogenic dyes (EtBr and Hoechst 33258). The results of HSA fluorescence study divulged static quenching of the complexes (1-4) with KSV values of 7.24×104 (1), 6.03×104 (2), 5.06×104 (3) and 2.85×104 (4) while Kb values; 1.16×105 (1), 2.01×104 (2), 5.84×103 (3) and 8.60×102 (4) suggested them potent avid binder of HSA. Additionally, comparative estimation of scavenging properties using DPPH, superoxide(O2.-), hydroxyl (OH-) and ABTS method and in-vitro cytotoxicity against different cell lines (MCF-7, HeLa and Hep G2) brought out distinct biopotency of complexes due to diverse structural features and chelation effect.

Synthesis, single-crystal, DNA interaction, spectrophotometric and spectroscopic characterization of the hydrogen-bonded charge transfer complex of 2-aminopyrimidine with π-acceptor chloranilic acid at different temperature in acetonitrile

The charge transfer (CT) interaction of 2-aminopyrimidine (AP) with chloranilic acid (CLA) as π-acceptor was investigated spectrophotometrically in acetonitrile at different temperatures in the range of 25-50°C. The 1:1 stoichiometry of the synthesized CT complex was detected using straight line method. Benesi-Hildebrand equation was used to determine the association constant (KCT), molar extinction coefficient (ε) and other physical parameters. Various thermodynamics parameters such as enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) were determined using UV-Visible spectrophotometry in acetonitrile at different temperatures. 1H NMR, FTIR, ESI-MS, elemental analyses, and UV-Visible techniques were used to characterize the hydrogen-bonded CT complex. 1H NMR spectroscopy was also used for the analysis of the CT complex where both hydrogen bond and charge transfer were present in its molecular composition. The interaction of the selected organic compound with Ct-DNA was well investigated using fluorescence spectroscopic method. Stern-Volmer constant (Ksv) was used to estimate the fluorescence quenching efficiency. Circular dichroism (CD) spectroscopy was employed to measure the conformational change of DNA in the presence of CT complex. Furthermore, the drug CT complex detected changes in its viscosity. The charge transfer complex was formed as a result of the transfer of the lone pair of electrons from donor to the acceptor and exhibits well resolved charge transfer bands in the regions where absorption by both donor and acceptor were absent. The thermal composition and stability of the CT complex were analyzed using thermogravimetric and differential thermal analysis (TGA and DTA) studies. The X-ray crystal structure was used for the interpretation of the structure of the [(AP)+ (CLA)-] CT complex. The crystal structure indicated that cation and anion are linked through strong N+H----O- type of hydrogen bond.