Identification of active-site residues of sheep liver serine hydroxymethyltransferase

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5'-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

Isoaccepting Lysine Transfer Ribonucleic Acid Species of Pseudomonas Aeruginosa

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide,three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAESephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNAL'y and a minor, tRNA'Ys. Co-chromatography of 14C-labelled tRNALYS and 3H-labelled tRNALy, on benzoylated DEAE-cellulose at pH4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,Gl) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.

Synthetic polypeptide with antifreeze activity

MUCH information has been gathered in recent years on the so-called 'antifreeze' proteins which lower the freezing point of the serum of certain marine fishes living in sub-zero water temperatures1−4. The proteins from the Antarctic fish Trematomus borchgrevinki are glycoproteins with a repeating alanyl-alanyl-threonyl tripeptide sequence, the threonyl residue being linked to a disaccharide1,2. In contrast, the antifreeze protein from the winter flounder Pseudopleuronectus americanus in the North American Atlantic coastal region is made up of eight ammo acids with no apparent repeating sequence of the residues and no sugar moiety (ref. 4 and unpublished work of C. L. Hew, C. C. Yip & G. Fletcher). The antifreeze activity of these proteins is not compatible with the known colligative properties of solutes in solution and the mechanism of their action is not yet fully understood. But a common feature of both types of the antifreeze proteins is the preponderance of alanine which accounts for over 60% of the total amino residues. This fact, together with the absence of the carbohydrate in the protein from the winter flounder, prompted us to attempt the synthesis of polypeptide analogues having comparable proportions of alanine in them along with suitable other amino acids. As a first step, we made use of the lack of any obvious periodicity in the distribution of the alanyl residues in the flounder's protein and attempted the synthesis of a random copolypeptide containing about 65 mol % of alanine and 35 mol % of aspartic acid. The choice of aspartic acid was made on the basis of its being the next major amino acid in the flounder's protein3,4 and on the expectation that its polar character will help the water-solubility of the alanine-rich copolypeptide, as in other studies on alanine-containing random copolymers. In addition, Duman and DeVries4 have earlier indicated the involvement of carboxyl groups on the antifreeze activity by chemical modification studies. We report here the synthesis of this polypeptide and show that it possesses antifreeze activity.

Diaryl dihydropyrazole-3-carboxamides with significant in vivo antiobesity activity related to CB1 receptor antagonism: Synthesis, biological evaluation, and molecular modeling in the homology model

A number of analogues of diaryl dihydropyrazole-3-carboxamides have been synthesized. Their activities were evaluated for appetite suppression and body weight reduction in animal models. Depending on the chemical modification of the selected dihydropyrazole scaffold, the lead compoundsthe bisulfate salt of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 26 and the bisulfate salt of (−)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 30showed significant body weight reduction in vivo, which is attributed to their CB1 antagonistic activity and exhibited a favorable pharmacokinetic profile. The molecular modeling studies also showed interactions of two isomers of (±)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 9 with CB1 receptor in the homology model similar to those of N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (rimonabant) 1 and 4S-(−)-3-(4-chlorophenyl)-N-methyl-N‘-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (SLV-319) 2.

Mapping of assembled epitopes with microgram quantities of antigen: Identification of an epitope at the receptor binding region of human follicle stimulating hormone

Identification of epitopes by modification studies has been reported by us recently. The method requires milligram quantities of antigen and since several proteins are not available in large quantities they are not amenable for such an investigation. One such protein is human follicle stimulating hormone (hFSH) whose mapping of epitopes is of importance in reproductive biology. Here we report a method that uses microgram quantities of hFSH to map a beta-specific epitope located at the receptor binding region. This identification has also been validated by the chemical modification method using heterologous antigen ovine follicle stimulating hormone (oFSH).

Tendency to promote ferroelectric distortion in Pr-modified PbTiO3

Structural, microstructural, and dielectric studies have been carried out on Pr-modified PbTiO3. A comparative analysis with La-modified PbTiO3 suggests that for chemical modification by same amount, the Pr-modified system has larger tetragonal strain and Curie point. No clear feature of relaxor ferroelectric state is observed for Pr concentration as high as x=0.35, suggesting that Pr modification is less effective, as compared to La-modification, in inducing a relaxor ferroelectric state. Results suggest that inspite of increased chemical disorder, Pr modification partly tends to restore the ferroelectric distortion of the lattice through partial occupancy of the Pr4+ ions on the Ti4+ sites.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Studies on a chitooligosaccharide-specific lectin from Coccinia indica. Thermodynamics and kinetics of umbelliferyl glycoside binding.

Coccinia indica agglutinin (CIA) is a chitooligosaccharide-specific lectin with two binding sites/homodimer of M(r) 32,000. Quenching studies implied tryptophan involvement in binding activity, which was confirmed by chemical modification experiments (A. R. Sanadi and A. Surolia, submitted for publication). Binding of 4-methylumbelliferyl chitooligosaccharides has been carried out to study their binding by CIA. Reversal experiments confirm the validity of the data previously obtained (A. R. Sanadi and A. Surolia, submitted for publication) from intrinsic fluorescence studies. Surprisingly, unlike wheat germ agglutinin, there is no consistent thermodynamic effect of the chromophoric label on binding activities as compared with the native sugars. From the changes in the optical properties of the chromophoric group upon binding to CIA, it has been possible to confirm that the tryptophan located in the binding site is closest to the fourth subsite. Thermodynamic analysis shows that the binding of the labeled tetrasaccharide is very strongly entropically driven, with the terminal, nonreducing sugar residue protruding from the binding pocket. The results of stopped-flow kinetic studies on the binding of the chromophoric trisaccharide by CIA show that the mechanism of binding is a one-step process.

Enzyme catalysed non-oxidative decarboxylation of aromatic acids II. Identification of active site residues of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger

In order to understand the mechanism of decarboxylation by 2,3-dihydroxybenzoic acid decarboxylase, chemical modification studies were carried out. Specific modification of the amino acid residues with diethylpyrocarbonate, N-bromosuccinimide and N-ethylmaleiimide revealed that at least one residue each of histidine, tryptophan and cysteine were essential for the activity. Various substrate analogs which were potential inhibitors significantly protected the enzyme against inactivation. The modification of residues at low concentration of the reagents and the protection experiments suggested that these amino acid residues might be present at the active site. Studies also suggested that the carboxyl and ortho-hydroxyl groups of the substrate are essential for interaction with the enzyme.

Interruptions of (CG)n sequences by GG, TG and CA need not prevent B to Z transition in solution

Oligonucleotides containing alternating purines-pyrimidines with AT base pairs have been shown to exist in the Z-form preferably in solid state. We report that oligodeoxyribonucleotides with GG, TG and CA interruptions in their alternating CG sequences can undergo B to Z transition in solution in the absence of any chemical modification or topological constraint. The sequences, d(CGCGCGGCGCGC) and d(CGTGCGCACG) have been synthesised and shown to adopt Z- conformation in presence of millimolar concentrations of Ni2+ under low water activity conditions. Significance of GG, TG and CA interruptions in the B to Z transition is discussed.

Fabrication of device quality films of high loaded PPy/MWCNT nanocomposites using pulsed laser deposition

Polypyrrole (PPy) - multiwalled carbonnanotubes (MWCNT) nanocomposites with various MWCNT loading were prepared by in situ inversion emulsion polymerization technique. High loading of the nano filler were evaluated because of available inherent high interface area for charge separation in the nanocomposites. Solution processing of these conducting polymer nanocomposites is difficult because, most of them are insoluble in organic solvents. Device quality films of these composites were prepared by using pulsed laser deposition techniques (PLD). Comparative study of X-ray photoelectron spectroscopy (XPS) of bulk and film show that there is no chemical modification of polymer on ablation with laser. TEM images indicate PPy layer on MWCNT surface. SEM micrographs indicate that the MWCNT's are distributed throughout the film. It was observed that MWCNT in the composite held together by polymer matrix. Further more MWCNT diameter does not change from bulk to film indicating that the polymer layer remains intact during ablation. Even for very high loadings (80 wt.% of MWCNT's) of nanocomposites device quality films were fabricated, indicating laser ablation is a suitable technique for fabrication of device quality films. Conductivity of both bulk and films were measured using collinear four point probe setup. It was found that overall conductivity increases with increase in MWCNT loading. Comparative study of thickness with conductivity indicates that maximum conductivity was observed around 0.2 mu m. (C) 2010 Elsevier B.V. All rights reserved.

Characterization of the zinc-metalloprotein nature of rat spermatidal protein TP2

Spermatidal transition protein, TP2, was purified from rat testes by Hg-affinity chromatography. The present study reports the details of the zinc-metalloprotein nature of TP2 by employing the Zn-65-blotting technique. Chemical modification of cysteine by iodoacetic acid, and histidine by diethylpyrocarbonate, resulted in a near complete inhibition of Zn-65-binding to TP2. The (65)Zinc-binding was localized to the V8 protease-derived N-terminal two-third polypeptide fragment. Circular dichroism spectroscopy studies of TP2 (zinc pre-incubated) and its V8 protease-derived polypeptide fragments revealed that the N-terminal fragment has a Type I-beta-turn spectrum, while the C-terminal fragment has a small but significant alpha-helical structure. EDTA altered the circular dichroism spectrum of TP2 and the N-terminal fragment (zinc binding domain) but not that of the C-terminal fragment.

Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA(1c) sample

Glycated hemoglobin (HbA(1c)) is a `gold standard' biomarker for assessing the glycemic index of an individual. HbA(1c) is formed due to nonenzymatic glycosylation at N-terminal valine residue of the P-globin chain. Cation exchange based high performance liquid chromatography (CE HPLC) is mostly used to quantify HbA(1c), in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE HPLC-based quantification,. resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA(1c) (52.1%) in the CE HPLC method. The observed HbA(1c) did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA(1c) and Hb Beckman might have resulted in the coelution of the variant with HbA(1c) in CE HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA(1c), it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA(1c) must be estimated using an alternative method. (C) 2015 Elsevier Inc. All rights reserved.

Luffa acutangula agglutinin: Primary structure determination and identification of a tryptophan residue involved in its carbohydrate-binding activity using mass spectrometry

A lectin from phloem exudates of Luffa acutangula (ridge gourd) was purified on chitin affinity chromatography and characterized for its amino acid sequence and to study the role of tryptophan in its activity. The purified lectin was subjected to various proteolytic digestions, and the resulting peptides were analyzed by liquid chromatography coupled electrospray ionization ion trap mass spectrometer. The peptide precursor ions were fragmented by collision-induced dissociation or electron transfer dissociation experiments, and a manual interpretation of MS/MS was performed to deduce amino acid sequence. This gave rise to almost complete sequence coverage of the lectin which showed high-sequence similarity with deduced sequences of phloem lectins present in the database. Chemical modification of lysine, tyrosine, histidine, arginine, aspartic acid, and glutamic acid residues did not inhibit the hemagglutinating activity. However, the modification of tryptophan residues using N-bromosuccinimide showed the loss of hemagglutinating activity. Additionally, the mapping of tryptophan residues was performed to determine the extent and number of residues modified, which revealed that six residues per molecule were oxidized suggesting their accessibility. The retention of the lectin activity was seen when the modifications were performed in the presence of chitooligosaccharides due to protection of a tryptophan residue (W-102) in the protein. These studies taken together have led to the identification of a particular tryptophan residue (W-102) in the activity of the lectin. (c) 2015 IUBMB Life, 67(12):943-953, 2015

Control of wettability of carbon nanotube array by reversible dry oxidation for superhydrophobic coating and supercapacitor applications

In this thesis, dry chemical modification methods involving UV/ozone, oxygen plasma, and vacuum annealing treatments are explored to precisely control the wettability of CNT arrays. By varying the exposure time of these treatments the surface concentration of oxygenated groups adsorbed on the CNT arrays can be controlled. CNT arrays with very low amount of oxygenated groups exhibit a superhydrophobic behavior. In addition to their extremely high static contact angle, they cannot be dispersed in DI water and their impedance in aqueous electrolytes is extremely high. These arrays have an extreme water repellency capability such that a water droplet will bounce off of their surface upon impact and a thin film of air is formed on their surface as they are immersed in a deep pool of water. In contrast, CNT arrays with very high surface concentration of oxygenated functional groups exhibit an extreme hydrophilic behavior. In addition to their extremely low static contact angle, they can be dispersed easily in DI water and their impedance in aqueous electrolytes is tremendously low. Since the bulk structure of the CNT arrays are preserved during the UV/ozone, oxygen plasma, and vacuum annealing treatments, all CNT arrays can be repeatedly switched between superhydrophilic and superhydrophobic, as long as their O/C ratio is kept below 18%.

The effect of oxidation using UV/ozone and oxygen plasma treatments is highly reversible as long as the O/C ratio of the CNT arrays is kept below 18%. At O/C ratios higher than 18%, the effect of oxidation is no longer reversible. This irreversible oxidation is caused by irreversible changes to the CNT atomic structure during the oxidation process. During the oxidation process, CNT arrays undergo three different processes. For CNT arrays with O/C ratios lower than 40%, the oxidation process results in the functionalization of CNT outer walls by oxygenated groups. Although this functionalization process introduces defects, vacancies and micropores opening, the graphitic structure of the CNT is still largely intact. For CNT arrays with O/C ratios between 40% and 45%, the oxidation process results in the etching of CNT outer walls. This etching process introduces large scale defects and holes that can be obviously seen under TEM at high magnification. Most of these holes are found to be several layers deep and, in some cases, a large portion of the CNT side walls are cut open. For CNT arrays with O/C ratios higher than 45%, the oxidation process results in the exfoliation of the CNT walls and amorphization of the remaining CNT structure. This amorphization process can be implied from the disappearance of C-C sp2 peak in the XPS spectra associated with the pi-bond network.

The impact behavior of water droplet impinging on superhydrophobic CNT arrays in a low viscosity regime is investigated for the first time. Here, the experimental data are presented in the form of several important impact behavior characteristics including critical Weber number, volume ratio, restitution coefficient, and maximum spreading diameter. As observed experimentally, three different impact regimes are identified while another impact regime is proposed. These regimes are partitioned by three critical Weber numbers, two of which are experimentally observed. The volume ratio between the primary and the secondary droplets is found to decrease with the increase of Weber number in all impact regimes other than the first one. In the first impact regime, this is found to be independent of Weber number since the droplet remains intact during and subsequent to the impingement. Experimental data show that the coefficient of restitution decreases with the increase of Weber number in all impact regimes. The rate of decrease of the coefficient of restitution in the high Weber number regime is found to be higher than that in the low and moderate Weber number. Experimental data also show that the maximum spreading factor increases with the increase of Weber number in all impact regimes. The rate of increase of the maximum spreading factor in the high Weber number regime is found to be higher than that in the low and moderate Weber number. Phenomenological approximations and interpretations of the experimental data, as well as brief comparisons to the previously proposed scaling laws, are shown here.

Dry oxidation methods are used for the first time to characterize the influence of oxidation on the capacitive behavior of CNT array EDLCs. The capacitive behavior of CNT array EDLCs can be tailored by varying their oxygen content, represented by their O/C ratio. The specific capacitance of these CNT arrays increases with the increase of their oxygen content in both KOH and Et4NBF4/PC electrolytes. As a result, their gravimetric energy density increases with the increase of their oxygen content. However, their gravimetric power density decreases with the increase of their oxygen content. The optimally oxidized CNT arrays are able to withstand more than 35,000 charge/discharge cycles in Et4NBF4/PC at a current density of 5 A/g while only losing 10% of their original capacitance.