Phenylglyoxal Research Articles

Comprehensive Assessment of Antioxidant, Antimicrobial, and Cytotoxic Activities of Schiff Bases Synthesized from Phenyl Glyoxal: Insights from Molecular Docking and ADMET Analysis

AbstractIn this study, novel Schiff base, PTIEO and PABHP were synthesized using Phenyl Glyoxal as a major constituent and their structures were elucidated through modern spectroscopic techniques. Antioxidant assays were performed using DPPH and ABTS assays. Further, PTIEO and PABHP were subjected to in vitro biological evaluations against ESKAP pathogens. This investigation revealed significant antibacterial activity of both compounds, with MIC below 64 μg mL−1 against Staphylococcus aureus and 64 μg mL−1 against Acinetobacter baummanii. In molecular docking studies, PTIEO shows enhanced binding energy with Acinetobacter baumannii bacterial protein Diaminopimelate epimerase at −7.45 Kcal mol−1, while PABHP exhibited −8.32 Kcal mol−1 binding energy with NADH‐dependent enoyl‐ACP reductase. Additionally, PTIEO and PABHP demonstrated good binding energy with Sortase‐A of Staphylococcus aureus at −7.59 Kcal mol−1 and −8.75 Kcal mol−1, respectively. Moreover, antifungal capabilities of these compounds were evaluated against pathogenic fungi Candida Albicans, Candida Parapsilosis, and Candida tropicalis, highlighting their broad‐spectrum antimicrobial properties. Moreover, PABHP displayed cytotoxicity with an IC50 value of 23.50 μg mL−1, suggesting its safer administration. ADMET calculations were also performed to demonstrate their effectiveness. Overall, these experiments attribute to the significant therapeutic behavior of Schiff bases as comprehensive biomedical agents.

A Facile Synthesis of Thiazole Derivatives bearing Imidazole Moiety, Schiff Bases and their O-Glucosides

A unique approach for the synthesis of new thiazole O-glycosides is presented in this work. 2-Amino-4-hydroxy-phenyl-1,3-thiazole-5-carboxaldehyde (3a) was reacted with phenyl glyoxal and benzil to form 4-(4-hydroxy-phenyl)-5-(4-phenyl-1H-imidazol-2-yl)-thiazol-2-amine (4a) and 4-(4-hydroxy-phenyl)-5-(4,5-diphenyl-1H-imidazol-2-yl)-thiazol-2-amine (4b), respectively. A series of substituted Schiff bases of 4a and 4b were synthesized reacting with various aryl aldehyde to form 2-(imino substituted benzal)-4-(4-hydroxy-phenyl)-5-(4-phenyl-1H-imidazol-2-yl)-thiazoles (5a-e) and 2-(imino substituted benzal)-4-(4-hydroxy-phenyl)-5-(4,5-diphenyl-1H-imidazol-2-yl)-thiazoles (5f-j). Glucosylation of compounds (5a-j) have been done by using acetobromoglucose as glucosyl donor to afford 2-(imino substituted benzal)-4-(2,3,4,6-tetra-O-acetyl-p-O-β-D-glucosidoxyphenyl)-5-(4-phenyl- 1H-imidazol-2-yl)-thiazoles (6a-e) and 2-(imino substituted benzal)-4-(2,3,4,6-tetra-O-acetyl-p-O-β- D-glucosidoxyphenyl)-5-(4,5-diphenyl-1H-imidazol-2-yl)-thiazoles (6f-j) further on deacetylation to produce 2-(imino substituted benzal)-4-(p-O-β-D-glucosidoxyphenyl)-5-(4-phenyl-1H-imidazol-2-yl)- thiazoles and 2-(imino substituted benzal)-4-(p-O-β-D-glucosidoxyphenyl)-5-(4,5-diphenyl-1Himidazole- 2-yl)-thiazoles (7f-j). The synthesized compounds were characterized by elemental analyses, FTIR, 1H & 13C NMR and electron mass spectra (EI-MS) techniques and then screened for their in vitro antimicrobial activity.

Iron(iii) chloride-catalyzed mechanochemical cascade synthesis of highly-substituted pyrrolyl indoles

Iron(iii) chloride has been found to serve as an efficient catalyst for a mechanochemical (ball milling) one-pot four- component cascade reaction of phenyl glyoxal, anilines, indoles and activated alkyne affording highly-substituted 3-(1H-pyrrol-2-yl)-1H-indoles. The procedure is beneficial because of mild conditions, easily available starting materials, cheap catalyst and possibility for scaling.

Chemiluminescence “signal-on-off” dual signals ratio biosensor based on single-stranded DNA functions as guy wires to detect EcoR V

Signal output mode is the important part of biosensor. In general, “signal on” and “signal off” are two common output modes. The development of dual signals-based ratio analysis as a powerful diagnostic tool has attracted widespread attention in the biosensor field in recent years. Dual signals ratio sensors with “signal on” and “signal off” are more favored because of their low background signal and better sensitivity and selectivity. In this study, inspired by the idea that EcoR V can cut specific sites of DNA to produce two corresponding fragments, and by using the capturing probe as guy wires, a reliable and sensitive method for EcoR V assay is developed based on the ratio of dual chemiluminescence (CL) signals for the first time. In particular, in the existence of the objective EcoR V, the substrate DNA would be degraded into two double stranded oligonucleotides with blunt ends which include the sequence I and the sequence II, then they can separately compete with two different corresponding capture probes on magnetic beads (MBs). One of capture probe hybridized with the sequence I containing more guanine (G) bases that reacted with the phenylglyoxal (PG) to produce chemical reaction which triggered a positive CL signal output I + CL as “signal-on”; another capture probe is priority to hybridize the sequence II, which triggered the weaker reporter DNA linked with horseradish peroxidase (HRP) probe to fall off the MBs, thereby outputting a negative CL signal I−CL as “signal-off”. By comparing the linear relation and the correlation coefficient, the I−CL/I + CL ratio method has better linear relation (0.01–10 U/mL) and higher sensitivity (0.0045 U/mL). In addition, this developed strategy of high selectivity which can directly detect low concentration of target EcoR V in human serum, and thus this dual ratio biosensor might offer a promising detection approach for clinical diagnostics.

A Fluorogenic Assay: Analysis of Chemical Modification of Lysine and Arginine to Control Proteolytic Activity of Trypsin

The chemical modification of amino acids plays an important role in the modulation of proteins or peptides and has useful applications in the activation and stabilization of enzymes, chemical biology, shotgun proteomics, and the production of peptide-based drugs. Although chemoselective modification of amino acids such as lysine and arginine via the insertion of respective chemical moieties as citraconic anhydride and phenyl glyoxal is important for achieving desired application objectives and has been extensively reported, the extent and chemoselectivity of the chemical modification of specific amino acids using specific chemical agents (blocking or modifying agents) has yet to be sufficiently clarified owing to a lack of suitable assay methodologies. In this study, we examined the utility of a fluorogenic assay method, based on a fluorogenic tripeptide substrate (FP-AA1-AA2-AA3) and the proteolytic enzyme trypsin, in determinations of the extent and chemoselectivity of the chemical modification of lysine or arginine. As substrates, we used two fluorogenic tripeptide probes, MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific substrate), which were designed, synthesized, and evaluated for chemoselective modification of specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in terms of half-maximal inhibitory concentrations (IC50) for the extent of modification and ratios of IC50 values (IC50arginine/IC50lysine and IC50lysine/IC50arginine) as a measure of the chemoselectivity of chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical modification.

Metabolic acclimation supports higher aluminium-induced secretion of citrate and malate in an aluminium-tolerant hybrid clone of Eucalyptus

BackgroundEucalyptus is the main plantation wood species, mostly grown in aluminized acid soils. To understand the response of Eucalyptus clones to aluminum (Al) toxicity, the Al-tolerant Eucalyptus grandis × E. urophylla clone GL-9 (designated “G9”) and the Al-sensitive E. urophylla clone GL-4 (designated “W4”) were employed to investigate the production and secretion of citrate and malate by roots.ResultsEucalyptus seedlings in hydroponics were exposed to the presence or absence of 4.4 mM Al at pH 4.0 for 24 h. The protein synthesis inhibitor cycloheximide (CHM) and anion channel blocker phenylglyoxal (PG) were applied to explore possible pathways involved in organic acid secretion. The secretion of malate and citrate was earlier and greater in G9 than in W4, corresponding to less Al accumulation in G9. The concentration of Al in G9 roots peaked after 1 h and decreased afterwards, corresponding with a rapid induction of malate secretion. A time-lag of about 6 h in citrate efflux in G9 was followed by robust secretion to support continuous Al-detoxification. Malate secretion alone may alleviate Al toxicity because the peaks of Al accumulation and malate secretion were simultaneous in W4, which did not secrete appreciable citrate. Enhanced activities of citrate synthase (CS) and phosphoenolpyruvate carboxylase (PEPC), and reduced activities of isocitrate dehydrogenase (IDH), aconitase (ACO) and malic enzyme (ME) were closely associated with the greater secretion of citrate in G9. PG effectively inhibited citrate and malate secretion in both Eucalyptus clones. CHM also inhibited malate and citrate secretion in G9, and citrate secretion in W4, but notably did not affect malate secretion in W4.ConclusionsG9 immediately secrete malate from roots, which had an initial effect on Al-detoxification, followed by time-delayed citrate secretion. Pre-existing anion channel protein first contributed to malate secretion, while synthesis of carrier protein appeared to be needed for citrate excretion. The changes of organic acid concentrations in response to Al can be achieved by enhanced CS and PEPC activities, but was supported by changes in the activities of other enzymes involved in organic acid metabolism. The above information may help to further explore genes related to Al-tolerance in Eucalyptus.

Bioinorganic Chemistry of Co (II) and Mn (II) Complexes

Objectives: Schiff base ligand 2-thio phenyl glyoxal anthranilic acid is used as the starting material for the synthesis of many biologically active compounds. The main objectives of the Study was to synthesize, characterization and biological evaluation of a octahedral complexes of Co (II) and Mn(II) containing 2- thiophenyl glyoxal anthranilic acid as ligand. Methods: Ethanolic solution of ligand 2-thiophenyl glyoxal anthranilic acid added into the ethanolic solution of metal acetate the resulting solution was refluxed for 2 hr the product thus obtained was filtered, washed with ethanol followed by ether and characterized by various spectral studies and biological significance. Results: The resulted complex was investigated by the help of elemental analysis, Molar Conductance, Magnetic moment, IR, Electronic and NMR Spectral Studies. Spectral data indicates that the geometry of the complexes are Octahedral. The result of biological and cytotoxic studies indicate that complexes are much more biologically active as compared to ligand fragments. Conclusion: The complexes are biologically active and were found to be effective in all the human cancer cell lines.

Construction of chemiluminescence aptasensor platform using magnetic microsphere for ochratoxin A detection based on G bases derivative reaction and Au NPs catalyzing luminol system

In this paper, two kinds of chemiluminescent (CL) aptasensors for detecting Ochratoxin A (OTA) were constructed using Magnetic microsphere (MBs) as separated carrier. For the label-free aptasensor, in presence of the OTA, the OTA aptamer specially bound with it, and this affinity function was stronger than hybridization interaction between OTA aptamer and the paired sequence (capture probe), and resulted in the quantitative reduction of aptamers that bound to the capture probe on the MBs. And thus, according to the CL signals changes which came from the transient reaction between guanine (G) in the OTA aptamer sequence and phenylglyoxal (PG), the OTA could be rapidly detected. Meanwhile, another Au nanoparticle (Au NPs) -labeled aptasensor based on the above-mentioned mechanism of aptamer affinitive substitution was also prepared, analytical target OTA could be sensitively determined with the CL signals which were generated by Au NPs catalyzing luminol chemiluminescence. Under the optimal conditions, the detection limits of two OTA sensors were 0.041 ng/mL and 0.0067 ng/mL, respectively. What is more, these two simple, convenient and low-cost strategies afford high sensitivity, specificity and suitability for detection of OTA in real samples.

Phenylglyoxal inhibition of the mitochondrial F1FO-ATPase activated by Mg2+ or by Ca2+ provides clues on the mitochondrial permeability transition pore

Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.

Characteristics of an Acidic Phytase from Aspergillus aculeatus APF1 for Dephytinization of Biofortified Wheat Genotypes.

Phytases are the special class of enzymes which have excellent application potential for enhancing the quality of food by decreasing its inherent anti-nutrient components. In current study, a protease-resistant, acidic phytase from Aspergillus aculeatus APF1 was partially purified by ammonium sulfate fractionation followed by chromatography techniques. The molecular weight of partially purified phytase was in range of 25-35kDa. The purified APF1 phytase was biochemically characterized and found catalytically active at pH3.0 and 50°C. The Km and Vmax values of APF1 phytase for calcium phytate were 3.21mM and 3.78U/mg protein, respectively. Variable activity was observed with metal ions and among inhibitors, chaotropic agents and organic solvents; phenyl glyoxal, potassium iodide, and butanol inhibited enzyme activity, respectively, while the enzyme activity was not majorly influenced by EDTA, urea, ethanol, and hexane. APF1 phytase treatment was found effective in dephytinization of flour biofortified wheat genotypes. Maximum decrease in phytic acid content was noticed in genotype MB-16-1-4 (89.98%) followed by PRH3-30-3 (82.32%) and PRH3-43-1 (81.47%). Overall, the study revealed that phytase from Aspergillus aculeatus APF1 could be effectively used in food and feed processing industry for enhancing nutritional value of food.

Extracellular lysine 38 plays a crucial role in pH-dependent transport via human monocarboxylate transporter 1

Human monocarboxylate transporters (hMCTs) are expressed in many tissues and mediate the transport of various substrates across the plasma membrane. Among hMCTs, hMCT1–4 cotransport H+ with monocarboxylates such as pyruvate and l-lactate, implying that these proteins recognize both substrate and H+. However, the mechanism of translocation, and particularly that of hMCT1 pH-dependent transport, remains largely unknown. This study aimed at identifying residues involved in the pH dependence of hMCT1 using a combination of amino acid-modifying reagents, site-directed mutagenesis in a Xenopus laevis oocyte expression system, and homology modeling. We showed that diethyl pyrocarbonate (DEPC), phenylglyoxal (PGO), and 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS), which react with histidine, arginine, and lysine residues respectively, all inhibited hMCT1 activity. Since DEPC, PGO, and DIDS are membrane impermeable reagents, we mutated to other residues individual histidine, arginine, and lysine residues located within the extracellular regions of hMCT1. Analyses of these mutants demonstrated that except for K38, the extracellular basic residues of hMCT1 were not involved in its transport activity and pH dependence. Moreover, analyses of various mutants in which K38 was substituted for another residue and of an hMCT1 homology model focusing on the location of K38 in the three-dimensional structure delineated the mechanism of hMCT1 pH dependence. Collectively, our data indicate that K38 plays an essential role in hMCT1 transport activity. We would like to propose a mechanism whereby K38 is positioned within a hydrophobic and narrow cavity that is part of the transport pathway, and regulates pH-dependent gating of hMCT1.

Arginine 107 of yeast ATP synthase subunit g mediates sensitivity of the mitochondrial permeability transition to phenylglyoxal

Modification with arginine-specific glyoxals modulates the permeability transition (PT) of rat liver mitochondria, with inhibitory or inducing effects that depend on the net charge of the adduct(s). Here, we show that phenylglyoxal (PGO) affects the PT in a species-specific manner (inhibition in mouse and yeast, induction in human and Drosophila mitochondria). Following the hypotheses (i) that the effects are mediated by conserved arginine(s) and (ii) that the PT is mediated by the F-ATP synthase, we have narrowed the search to 60 arginines. Most of these residues are located in subunits α, β, γ, ϵ, a, and c and were excluded because PGO modification did not significantly affect enzyme catalysis. On the other hand, yeast mitochondria lacking subunit g or bearing a subunit g R107A mutation were totally resistant to PT inhibition by PGO. Thus, the effect of PGO on the PT is specifically mediated by Arg-107, the only subunit g arginine that has been conserved across species. These findings are evidence that the PT is mediated by F-ATP synthase.

Molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould Sporotrichum thermophile reveals insights into molecular catalysis and biochemical properties

A thermostable and protease-resistant HAP-phytase of Sporotrichum thermophile was over-expressed in Pichia pastoris X-33. Purified recombinant phytase displayed all its biochemical properties similar to wild type. Molecular modeling and docking of phytase with various substrates showed differential binding patterns with GoldScore values ranging from 40.61 to 79.78. Docking with different substrates revealed strong binding affinity with ATP and phytic acid, while the lowest with AMP and phosphoenol pyruvate. This was further confirmed using biochemical assays, as the recombinant enzyme displayed broad substrate specificity. Docking with inhibitors also showed differential binding with GoldScore values ranging from 22.94 (2,3-butanedione) to 85.72 (myo-inositol hexasulphate). Validation using biochemical analysis revealed that both 2,3-butanedione and phenyl glyoxal inhibited the phytase activity significantly. Furthermore, presence of inorganic phosphate in the reaction mixture also inhibited the phytase activity, as there was no activity at and beyond 0.8 mM. Docking of phytase with metavanadate showed binding at the same atom in the active-site where the substrate i.e. phytic acid binds. Vanadium incorporation resulted in the catalytic conversion of phytase into a peroxidase with concomitant inhibition of phytase activity. Peroxidase activity was high in acidic range and the product formation showed correlation with reaction time. Furthermore, molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould S. thermophile reveals insights into molecular catalysis that is validated by the biochemical properties.

ChemInform Abstract: Peroxide‐Free Pd(II)‐Catalyzed ortho Aroylation and ortho Halogenation of Directing Arenes.

A Pd(II)-catalyzed peroxide-free ortho aroylation of directing arenes has been developed via cross dehydrogenative coupling (CDC) in the presence of the terminal oxidant Cu(OAc)2·H2O. Ortho aroylation of directing arenes proceeds via decarbonylation of the in situ generated phenyl glyoxal, which is obtained from 2-acetoxyacetophenone in the presence of the oxidant Cu(OAc)2·H2O. However, changing the oxidant to CuX2 (X = Cl, Br) provided exclusive di-ortho-halogenated 2-arylbenzothiazoles. During the halogenation, CuX2 served the dual role of a halogen source as well as a co-oxidant.

Label-free chemiluminescent strategy for highly selective and sensitive detection of adenosine triphosphate by cofactor-dependent enzymatic ligation-triggered polymerase chain reaction

In this work, a novel label-free chemiluminescent (CL) assay system was developed for highly sensitive and selective detection of adenosine triphosphate (ATP). The strategy relies on the powerful signal amplification capability of rolling circle amplification (RCA), the preferential binding ability of graphene oxide (GO) to single-stranded DNA (ssDNA), and instantaneous derivatization reaction between phenylglyoxal (PGO) and guanine nucleobases (G). In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The formed long single-stranded DNA which contained an amount of guanine bases could be adsorbed on the surface of GO forming DNA-GO complexes. And the CL signal of DNA-GO complexes can be obtained via the instantaneous derivatization reaction between PGO and guanine bases. The CL signal increased linearly with the concentration of ATP from 0.1 to 2.5nM with a detection limit of 0.03nM. The system also showed high specificity to ATP against its analogues such as CTP, GTP, UTP, AMP, ADP, BSA and CaM. In addition, ATP has been determined in diluted serum indicating the applicability of this assay.

Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry

Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence.We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications.

Peroxide-Free Pd(II)-Catalyzed Ortho Aroylation and Ortho Halogenation of Directing Arenes.

A Pd(II)-catalyzed peroxide-free ortho aroylation of directing arenes has been developed via cross dehydrogenative coupling (CDC) in the presence of the terminal oxidant Cu(OAc)2·H2O. Ortho aroylation of directing arenes proceeds via decarbonylation of the in situ generated phenyl glyoxal, which is obtained from 2-acetoxyacetophenone in the presence of the oxidant Cu(OAc)2·H2O. However, changing the oxidant to CuX2 (X = Cl, Br) provided exclusive di-ortho-halogenated 2-arylbenzothiazoles. During the halogenation, CuX2 served the dual role of a halogen source as well as a co-oxidant.

ChemInform Abstract: Ultrasound‐Mediated One‐Pot Synthesis of 7‐Methyl‐Substituted Pyrido[4,3‐d]pyrimidine Scaffolds by a Catalyst‐Free Protocol.

AbstractThe reaction succeeds using aryl aldehydes as the reaction partner, but fails with alkyl aldehydes and phenyl glyoxal derivatives.

Catalytic Performance of Brønsted and Lewis Acid Sites in Phenylglyoxal Conversion on Flame‐Derived Silica–Zirconia

AbstractFlame‐derived silica–zirconia has been used for the promising one‐step catalytic conversion of phenylglyoxal (PG) to mandelates, important intermediates in pharmacy and fine chemistry. In the literature it was proposed that Lewis acid sites (LAS) on solid acids promote mandelate production, whereas Brønsted acid sites (BAS) only generate acetal byproducts during PG conversion. Herein it is shown that ZrO2, which contains only LAS and no BAS, exhibits a very low activity for PG conversion to afford a yield of mandelate of only 8 % after 6 h. The activity of this ZrO2 catalyst was confirmed by the condensation of acetone, in which its LAS could activate the carbonyl groups immediately for the fast condensation of all acetone molecules. The introduction of BAS by the admixing of silica precursor to the feed in the flame‐spray pyrolysis enhanced the yield of mandelate up to 52–67 %. This indicates that BAS are mainly responsible for the PG conversion to mandelate on silica–zirconia catalysts and that their LAS are nearly inactive for this reaction.

Tryptophan environment and functional characterization of a kinetically stable serine protease containing a polyproline II fold.

The single tryptophan residue from Nocardiopsis sp. serine protease (NprotI) was studied for its microenvironment using steady state and time-resolved fluorescence. The emission maximum was observed at 353nm with excitation at 295nm indicating tryptophan to be solvent exposed. Upon denaturation with 6M guanidinum thiocyanate (GuSCN) the emission maxima was shifted to 360nm. Solute quenching studies were performed with neutral (acrylamide) and ionic (I(-) and Cs(+)) quenchers to probe the exposure and accessibility of tryptophan residue of the protein. Maximum quenching was observed with acrylamide. In the native state, quenching was not observed with Cs(+) indicating presence of only positively charged environment surrounding tryptophan. However; in denatured protein, quenching was observed with Cs(+), indicating charge reorientation after denaturation. No quenching was observed with Cs(+) even at pH1.0 or 10.0; while at acidic pH, a higher rate of quenching was observed with KI. This indicated presence of more positive charge surrounding tryptophan at acidic pH. In time resolved fluorescence measurements, the fluorescence decay curves could be best fitted to monoexponential pattern with lifetimes of 5.13ns for NprotI indicating one conformer of the trp. Chemical modification studies with phenyl glyoxal suggested presence of Arg near the active site of the enzyme. No inhibition was seen with soyabean trypsin and limabean inhibitors, while, CanPI uncompetitively inhibited NprotI. Various salts from Hofmeister series were shown to decrease the activity and PPII content of NprotI.