Effect of Acetate and Carbonate Buffers on the Photolysis of Riboflavin in Aqueous Solution: A Kinetic Study

The photolysis of riboflavin (RF) in the presence of acetate buffer (pH 3.8–5.6) and carbonate buffer (pH 9.2–10.8) has been studied using a multicomponent spectrophotometric method for the simultaneous assay of RF and its photoproducts. Acetate and carbonate buffers have been found to catalyze the photolysis reaction of RF. The apparent first-order rate constants for the acetate-catalyzed reaction range from 0.20 to 2.86 × 10⁻⁴ s⁻¹ and for the carbonate-catalyzed reaction from 3.33 to 15.89 × 10⁻⁴ s⁻¹. The second-order rate constants for the interaction of RF with the acetate and the carbonate ions range from 2.04 to 4.33 × 10⁻⁴ M⁻¹ s⁻¹ and from 3.71 to 11.80 × 10⁻⁴ M⁻¹ s⁻¹, respectively. The k-pH profile for the acetate-catalyzed reaction is bell shaped and for the carbonate-catalyzed reaction a steep curve. Both HCO₃⁻ and CO₃^2 − ions are involved in the catalysis of the photolysis reaction in alkaline solution. The rate constants for the HCO₃⁻ and CO₃^2 − ions catalyzed reactions are 0.72 and 1.38 × 10⁻³ M⁻¹ s⁻¹, respectively, indicating a major role of CO₃^2 − ions in the catalysis reaction. The loss of RF fluorescence in acetate buffer suggests an interaction between RF and acetate ions to promote the photolysis reaction. The optimum stability of RF solutions is observed in the pH range 5–6, which is suitable for pharmaceutical preparations.KEY WORDS: acetate effect, carbonate effect, photolysis, riboflavin, spectrophotometric assay     

A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.     

Predicted binding of certain antifilarial compounds with glutathione-S-transferase of human Filariids

Glutathione-S-transferase is a major phase-II detoxification enzyme in parasitic helminthes. Previous research highlights the importance of GSTs in the establishment of chronic infections in cytotoxic microenvironments. Filarial nematodes depend on these detoxification enzymes for their survival in the host. GST plays an important role in filariasis and other diseases. GST from W.bancrofti and B.malayi are very much different from human GST. This structural difference makes GST potential chemotherapeutic targets for antifilarial treatment. In this study we have checked the efficacy of some well known antifilarial compounds against GST from B.malayi and W.bancrofti. The structure of BmGST was modeled using modeller9v10 and was submitted to PMDB. Molecular docking study reveals arbindazole to be the most potent compounds against GST from both the filarial parasites. Role of some residues playing important role in the binding of compounds within the active site of GST has also been revealed in the present study. The BmGST and WbGST structural information and docking studies could aid in screening new antifilarials or selective inhibitors for chemotherapy against filariasis.

Abbreviations

GST - Glutathione-S-transferase, Bm - Brugia malayi, Wb - Wuchereria bancrofti.     

Conformation and thermodynamic stability of pre-molten and molten globule states of mammalian cytochromes-c

Proteins in cells fold via a number of intermediates. These intermediates are quite important as they guide the protein to attain its unique native conformation. To solve the immensely difficult problem of protein folding, it is necessary to characterize intermediates which will unravel the mystery of the steps involved in the proper folding of proteins. Cytochromes-c (cyts-c) have played an important role in studies of the earliest events and intermediates in protein folding. They have always been considered as model proteins for protein folding studies due to their intrinsic properties that can be measured by multiple probes. A large number of different solvent conditions have been employed to obtain equilibrium intermediates of cyts-c. These intermediates show structural heterogeneity which is mainly due to the different solvent conditions used to induce them. In this review we present results of conformational and thermodynamic characterization of equilibrium intermediates (molten globules and pre-molten globules) of the mammalian cyts-c under different solvent conditions.     

Phosphoproteome sequence analysis and significance: Mining association patterns around phosphorylation sites utilizing MAPRes

Phosphorylation, one of the most common protein post‐translational modifications (PTMs) on hydroxyl groups of S/T/Y is catalyzed by kinases and involves the presence or absence of certain amino acid residues in the vicinity of the phosphorylation sites. Using MAPRes, we have analyzed the substrate proteins of Phospho.ELM 7.0 and found that there are both general and specific requirements for the presence or absence of particular amino acids in the vicinity of phosphorylated S/T/Y for both of the phosphorylation data, whether or not kinase information was taken into account. Patterns extracted by MAPRes for kinase‐specific data have been utilized to find the consensus sequence motifs for various kinases required to catalyze the process of phosphorylation on S/T/Y. These consensus sequences for different kinase groups, families, and individual members are consistent with those described earlier with some novel consensus reported for the first time. A comparison study for the patterns mined by MAPRes with the results of existing prediction methods was performed by searching for these patterns in the vicinity of phosphorylation sites predicted by different available method. This comparison resulted in 87–98% conformity with the results of the predictions by available methods. Additionally, the patterns mined by MAPRes for substrate sites included 61 kinases, the highest number analyzed so far. J. Cell. Biochem. 108: 64–74, 2009. © 2009 Wiley‐Liss, Inc.     

A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0

Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8-naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 °C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural characteristics of a pre-molten-globule state at pH 2 and 25 °C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 °C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein.     

As(V) Reduction,As(III) Oxidation,and Cr(VI) Reduction by Multi-metal-resistant Bacillus subtilis,Bacillus safensis,and Bacillus cereus Species Isolated from Wastewater Treatment Plant

Industrial progress has resulted in threatening concentrations of toxic metals in various areas of the world. Bioremediation is an economical alternative to chemical methods. Bacteria resistant to As(III), As(V), Cr, Co, Cu, Cd, Hg, Ni, Pb, Se, and Zn were isolated from the wastewater treatment plant of Kasur, Pakistan. Highest resistance against all metals was exhibited by MX-1, MX-3, MX-4, and MX-5. The isolates possessed dual ability to oxidize as well as to reduce As. Highest As(V) reduction (454 µM) was exhibited by MX-1, while most As(III) oxidation was shown by MX-3 (170 µM). The isolates were also capable of reducing Cr(VI), and maximum Cr(VI) reduction (500 µM) was exhibited by MX-3. Transformation of DH5α with MX-1 plasmid showed that resistance genes for As(III), As(V), Cr, Cd, Se, Hg, and Ni were plasmid borne, while in case of MX-3, resistance genes for As(III), As(V), Co, Cu, Se, Pb, Zn, and Ni were present on plasmid. MX-1 and MX-3 were also positive for auxin production (37 and 32.96 µg ml^?1, respectively). MX-3 was also found to produce hydrogen cyanide (HCN) and solubilized phosphate. These isolates promoted plant (Vigna radiata) growth both in the presence and absence of the metals. MX-1, MX-3, and MX-5 were identified as Bacillus subtilis, Bacillus safensis, and Bacillus cereus through 16S rRNA gene sequencing, respectively. Such bacteria having multiple traits of resisting multiple metals, dual ability to oxidize/reduce As, and reduce Cr(VI) along with the ability to support plant growth are good tools for remediation of metal-contaminated sites and its cultivation.     

Phytochemical and Biological Activities of Pseudocalymma elegans: A False Garlic

Evaluation of phytochemical constituents and antioxidant and antimicrobial activities of hexane (PELH), dichloromethane (PELDCM), ethyl acetate (PELEA), and MeOH (PELM) extracts of young leaves of Pseudocalymma elegans have been carried out. Moreover, extracts have also been explored for the presence of sulphur containing compounds, 1,2‐dithiolane ( 33 ), diallyl disulfide ( 35 ), 3‐vinyl‐1,2‐dithiacyclohex‐5‐ene ( 37 ), and diallyl trisulfide ( 38 ) responsible for the garlic like smell of P. elegans. All the extracts were found to be antioxidant and showed potent inhibition with IC₅₀ values of 0.168 ± 0.001, 0.128 ± 0.002, 0.221 ± 0.011, and 0.054 ± 0.001, respectively, as compared to standard drugs ascorbic acid (AA) and butylated hydroxytoluene (BHT). The ethyl acetate extract (PELE) showed excellent activities against few Gram‐positive and Gram‐negative bacteria and some fungi as compared with standard drug ceftriaxone (3rd generation cephalosporin) and nystatin, respectively. Chemical constituents of hexane, dichloromethane, and ethyl acetate extracts were identified by gas chromatography‐mass spectrometry and mass spectral library search. Over all 55 chemical constituents were first time identified from the leaves which included branched and n‐hydrocarbons, fatty acids, fatty acid methyl esters, fatty alcohols, terpenes, alkaloid, vitamins, glycosides, aromatic compounds, and sulfur containing compounds. Two known chemical constituents, ursolic acid ( 1 ) and β‐amyrin ( 2 ), were also purified for the first time from the MeOH extract. To elucidate the structures of these compounds, UV, IR, EI‐MS, ¹H‐ and ¹³C‐NMR spectroscopy were used.