Cleavage Of Bovine Serum Albumin Research Articles

Extracellular BSA-degrading SAPs in the rare pathogen Meyerozyma guilliermondii strain SO as potential virulence factors in candidiasis

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects.

An optimization by response surface methodology for the enhanced production of rMBSP from Pichia pastoris and study of its application

Background: Recombinant myofibril-bound serine proteinase (rMBSP) was successfully expressed in Pichia pastoris GS115 in our laboratory. However, low production of rMBSP in shake flask constraints further exploration of properties. Methods: A 5-L high cell density fermentation was performed and the fermentation medium was optimized. Response surface methodology (RSM) was used to optimize the culture condition through modeling three selected parameter. Results: Under the optimized culture medium (LBSM, 1% yeast powder and 1% peptone) and culture conditions (induction pH 5.5, temperature 29 °C, time 40 h), the yield of rMBSP was 420 mg/L in a 5-L fermenter, which was a 6-fold increase over thar, expressed in flask cultivation. The desired enzyme was purified by two-step, which yielded a 33.7% recovery of a product that had over 85% purity. The activity of purified rMBSP was significantly inhibited by Ca2+, Mg2+, SDS, guanidine hydrochloeide, acetone, isopropanol, chloroform, n-hexane and n-heptane. Enzymatic analysis revealed a Km of 2.89 ± 0.09 μM and a Vmax of 14.20 ± 0.12 nM•min−1 for rMBSP. LC-MS/MS analysis demonstrated the specific cleavage of bovine serum albumin by rMPSP. Conclusion: These findings suggest that rMPSP has potential as a valuable enzyme for protein science research.

Photosensitive and pH-dependent activity of pyrazine-functionalized carbazole derivative as promising antifungal and imaging agent

Carbazole skeleton plays a significant role as a structural scaffold of many pharmacologically active compounds. Pyrazine-functionalized carbazole derivative was constructed by coupling 2-amino-5-bromo-3-methylaminepyrazine (ABMAP) into 3 and 6 positions of the carbazole ring. Multi-experimental methods were used, e.g., potentiometric, spectroscopic (ATR, UV, XRD powder,1H and13C NMR), electrochemical (cyclic voltammetry), and optical techniques, to receive the complete structural analysis, physicochemical (pKa, logP) and biological profile of a new carbazole derivative with acronym 3,6-PIRAMICAR. The interaction ability of the compound studied with potential cellular targets like Calf Thymus DNA (CT-DNA), or Bovine Serum Albumin (BSA) were also taken into account. Experiments showed the existence of strong binding, but no DNA or BSA cleavage was observed. The comparative analyzes of compounds anti-Candida action clearly show pH-dependent antifungal activity of 3,6-PIRAMICAR, which was strongly stimulated in the acidic media. Surprisingly, the titled compound turn out to be much more effective (14 times by MIC50; 8 times by MIC; c.a. 4 times by MFC) against Candida krusei than fluconazole at pH 4.

Label-free fluorescence turn-on strategy for trypsin activity based on thiolate-protected gold nanoclusters with bovine serum albumin as the substrate

By using bovine serum albumin (BSA) as the enzyme substrate, we propose a label-free, convenient, reliable and highly sensitive fluorescence “turn-on” assay for trypsin activity and inhibitor screening by means of fluorescent gold nanoclusters capped with 11-mercaptoundecanoic acid (namely AuNCs@11-MUA) for the first time. The principle for the assay depends on the following features: (1) Cu2+-triggered fluorescence quenching of AuNCs owing to the binding between Cu2+ and the carboxyl groups, (2) the cleavage of BSA into amino acids/peptide fragments catalyzed by trypsin, and (3) the stronger binding ability between amino acids/peptide fragments and Cu2+ than that between AuNCs and Cu2+, accompanied by the conversion from fluorescence off-state to on-state. Under the optimized conditions, the proposed sensing assay shows a good linear relationship from 0.01 to 2μg/mL and provides an inspiring detection limit of 0.004μg/mL, which possesses adequate sensitivity for practical samples of human urine. In addition, our proposed trypsin bioassay can be expanded into the inhibitor evaluation by using trypsin inhibitor from soybean as a model. Such unprecedented AuNCs-based fluorescence “turn-on” analytical proposal for assessing trypsin activity gives a new sight to develop several other potential bioassays or enzyme activity assays with the help of different kinds of substrates in the near future.

Evaluation of the cell cytotoxicity and DNA/BSA binding and cleavage activity of some dioxidovanadium(V) complexes containing aroylhydrazones

Three dioxidovanadium(V) complexes [VO2L1–3] (1–3) [HL1=1-napthoyl hydrazone of 2-acetyl pyridine, HL2=2-furoyl hydrazone of 2-acetyl pyridine and H2L3=isonicotinoyl hydrazone of 2-hydroxy benzaldehyde] have been reported. All the complexes were characterized by various spectroscopy (IR, UV-visible and NMR) and the molecular structures of 1 and 2 were characterized by single crystal X-ray diffraction technique. Structural report established five-coordinate geometries, distorted toward square pyramidal for each of 1 and 2, based on a tridentate -O,N,N coordinating anion and two oxido–O atoms. The experimental results show that the complexes interact with calf–thymus DNA (CT-DNA) possibly by a groove binding mode, with binding constants of ~105M−1. All complexes show good photo-induced cleavage of pUC19 supercoiled plasmid DNA with complex 1 showing the highest photo-induced DNA cleavage activity of ~68%. 1–3 also exhibit moderate binding affinity in the range of 103–104M−1 towards bovine serum albumin (BSA), while all the complexes show good photo-induced BSA cleavage activity. Moreover the antiproliferative activity of all these complexes was studied, which reveal all compounds are significantly cytotoxic towards the HeLa cell line.

Dissymmetric thiosemicarbazone ligands containing substituted aldehyde arm and their ruthenium(II) carbonyl complexes with PPh3/AsPh3 as ancillary ligands: Synthesis, structural characterization, DNA/BSA interaction and in vitro anticancer activity

A serious of new dissymmetric ruthenium(II) carbonyl complexes of the type [Ru(CO)(EPh3)(L1–2)] (1–4), [E = P or As; L1 = N4-(2-Hydroxy-5-chlorobenzylidene)-2-amino-5-chlorobenzophenone thiosemicarbazone; L2 = N4-(2-Hydroxynaphthalene-1-carbaldehyde)-2-amino-5-chlorobenzophenone thiosemicarbazone] have been synthesized and characterized by several spectroscopic studies. The molecular structure of the ligand L1 and the ruthenium(II) carbonyl complexes (2, 4) have been analyzed by single crystal X-ray studies, and found that the ruthenium(II) complexes possess a distorted octahedral geometry. The DNA binding studies such as emissive titration, Ethidium bromide/Methylene blue (EB/MB) displacement assay and viscometry measurements revealed that the ruthenium(II) complexes bound with calf thymus DNA through intercalative mode with relatively high binding constant values. Further, the interactions of the complexes with bovine serum albumin (BSA) were also investigated using fluorescence spectroscopic methods, which showed that the new complexes could bind strongly with BSA. The complexes (1–4) were tested for DNA and BSA cleavage activities, and the results showed that the complexes exhibited good cleavage properties. In addition, the newly synthesized ruthenium(II) complexes possess better in vitro cytotoxic activities against various cell lines (MCF-7, Hop62, MDA-MB-435) and AO/EB staining method showed that these complexes induced apoptosis of MCF-7 cell lines.

Nonspecific cleavage of proteins using graphene oxide

In this article, we report the intrinsic catalytic activity of graphene oxide (GO) for the nonspecific cleavage of proteins. We used bovine serum albumin (BSA) and a recombinant esterase (rEstKp) from the cold-adapted bacterium Pseudomonas mandelii as test proteins. Cleavage of BSA and rEstKp was nonspecific regarding amino acid sequence, but it exhibited dependence on temperature, time, and the amount of GO. However, cleavage of the proteins did not result in complete hydrolysis into their constituent amino acids. GO also invoked hydrolysis of p-nitrophenyl esters at moderate temperatures lower than those required for peptide hydrolysis regardless of chain length of the fatty acyl esters. Based on the results, the functional groups of GO, including alcohols, phenols, and carboxylates, can be considered as crucial roles in the GO-mediated hydrolysis of peptides and esters via general acid–base catalysis. Our findings provide novel insights into the role of GO as a carbocatalyst with nonspecific endopeptidase activity in biochemical reactions.

Cysteine protease is a major component in the excretory/secretory products of Euclinostomum heterostomum (Digenea: Clinostomidae)

Cysteine proteases of parasite organisms play numerous indispensable roles in tissue penetration, feeding, immunoevasion, virulence, egg hatching and metacercarial excystment. They are critical key enzymes in the biology of parasites and have been exploited as serodiagnostic markers, therapeutic and vaccine targets. In the present study, the cysteine proteases in the in vitro released excretory/secretory (E/S) products of the digenetic trematode parasite, Euclinostomum heterostomum have been analysed. The encysted progenetic metacercariae of E. heterostomum collected from the infected liver and kidney of Channa punctatus were excysted in vitro and incubated in phosphate buffer at 37 ± 1 °C, and the E/S products released were analysed. The spectrophotometric analysis of the proteases revealed active hydrolysis of chromogenic substrate, azocoll, in a time-, temperature- and pH-dependent manner. Optimum activity was observed at pH 7.0 at 37 ± 1 °C, and with 1 mM each of various protease inhibitors (Mini Protease Inhibitor Cocktail, ethylene diaminetetraacetic acid, phenyl methyl sulphonyl fluoride, iodoacetamide and 1,10-phenanthroline) used, significant inhibition was observed by iodoacetamide and 85% of inhibition at a concentration of 2 mM, suggesting that cysteine protease is a major component in the E/S of this parasite. Four discrete protease bands of Mr 36, 39, 43 and 47 kDa were identified by gelatin-substrate zymography. Maximum gelatinolytic activity was observed at pH 7.0, and among various inhibitors used, almost complete disappearance of protease bands was observed by 2 mM iodoacetamide. The proteolytic cleavage of bovine serum albumin, bovine haemoglobin and human haemoglobin in vitro were also studied.

Soil proteomics: An assessment of its potential for archaeological site interpretation

In this work, two sets of experiments were carried out to assess the potential of soil proteomics for archaeological site interpretation. First, we examined the effects of various protein isolation reagents and soil constituents on peptide mass fingerprinting (PMF) of soil-like materials spiked with bovine serum albumin (BSA). In a subsequent case study, we assessed the relative age of soils from an ancient clay floor of a Roman farmhouse using amino acid racemization and then applied MALDI-TOF–MS–MS to detect and identify biomarkers for human occupation. The results from the first experiments indicate that BSA isolation efficiencies are hampered by the presence of clays and reactive organic matter (glucose), whereas these effects are, to some extent, reversed by the presence of humic acids. We also show that specific regions of the BSA molecule (the out most α-helical regions of domain 2 and 3) are more susceptible to isolation than other regions and this suggest that soil proteins can be only partly isolated. Soil–protein interactions were also found to inhibit tryptic cleavage of BSA, resulting in an enhanced specificity of BSA peptides. Our results further stress the importance of multiple or sequential protein isolation protocols with a focus on citrate and hydrofluoric acid. Results from the case study indicate that the ancient floor samples contain degraded and most likely aged proteinaceous matter, probably of keratinaceous origin.

Cytotoxic 1,2-Dialkynylimidazole-Based Aza-Enediynes: Aza-Bergman Rearrangement Rates Do Not Predict Cytotoxicity

A new class of potential antitumor agents inspired by the enediyne antitumor antibiotics has been synthesized: the 1,2-dialkynylimidazoles. The aza-Bergman rearrangement of these 1,2-dialkynylimidazoles has been investigated theoretically at the B3LYP/6-31G(d,p) level and experimentally by measuring the kinetics of rearrangement in 1,4-cyclohexadiene. There is a good correlation between the theoretical and experimental results; subtle substituent effects on the initial aza-Bergman cyclization barrier predicted by theory are confirmed by experiment. Yet, despite the ability of these 1,2-dialkynylimidazoles to undergo Bergman rearrangement to diradical/carbene intermediates under relatively mild conditions, there is no correlation between the rate of Bergman cyclization and cytotoxicity to A459 cells. In addition, cytotoxic 1,2-dialkynylimidazoles do not cause nicking of supercoiled plasmid DNA or cleavage of bovine serum albumin. An alternative mechanism for cytotoxicity involving the unexpected selective thiol addition to the N-ethynyl group of certain 1,2-dialkynylimidazoles is proposed.

Applications of the time-resolved two-colour two-photon excitation of UV fluorophores using femtosecond laser pulses

A short overview of the principles and applications of the two-colour two-photon (2C2P) excitation of fluorescence by using femtosecond pulses is given. Fluorescence is generated by the simultaneous absorption of an 800 nm photon and a 400 nm photon of overlapping laser beams of a titanium:sapphire laser. Two examples of its application are presented: firstly, it is used to monitor the enzymatic cleavage of bovine serum albumin (BSA) by elastase. The fluorescent amino acid tryptophan present in BSA is excited corresponding to an effective one-photon wavelength of 266 nm. Secondly, it is shown how one can utilize the different polarizations of the excited beams for determining the symmetry of the excited states of molecules, exemplarily shown for p-terphenyl in cyclohexane. Further applications and experiments for 2C2P are suggested for using it in UV-fluorescence microscopy and for determining the properties of the electronic states of biomolecules by using differently polarized photons.

Photocytotoxic 3d-Metal Scorpionates with a 1,8-Naphthalimide Chromophore Showing Photoinduced DNA and Protein Cleavage Activity

Ternary 3d-metal complexes of formulation [M(Tp(Ph))(py-nap)](ClO(4)) (1-3), where M is Co(II) (1), Cu(II) (2), and Zn(II) (3); Tp(Ph) is anionic tris(3-phenylpyrazolyl)borate; and py-nap is a pyridyl ligand with a conjugated 1,8-naphthalimide moiety, have been prepared from the reaction of metal perchlorate with KTp(Ph) and py-nap in CH(2)Cl(2). The complexes have been characterized from analytical and physicochemical data. The complexes are stable in solution as evidenced from the electrospray ionization mass spectrometry data. The complexes show good binding propensity with calf thymus (CT) DNA, giving binding constant (K(b)) values of approximately 10(5) M(-1) and a molecular "light-switch" effect that results in an enhancement of the emission intensity of the naphthalimide chromophore on binding to CT DNA. The complexes do not show any hydrolytic cleavage of DNA. They show poor chemical nuclease activity in the presence of 3-mercaptopropionic acid or hydrogen peroxide (H(2)O(2)). The Co(II) and Cu(II) complexes exhibit oxidative pUC19 DNA cleavage activity in UV-A light of 365 nm. The Zn(II) complex shows moderate DNA photocleavage activity at 365 nm. The Cu(II) complex 2 displays photoinduced DNA cleavage activity in red light of 647.1 nm and 676 nm and near-IR light of >750 nm. A mechanistic study in UV-A and visible light suggests the involvement of the hydroxyl radical as the reactive species in the DNA photocleavage reactions. The complexes also show good bovine serum albumin (BSA) protein binding propensity, giving K(BSA) values of approximately 10(5) M(-1). Complexes 1 and 2 display significant photoinduced BSA cleavage activity in UV-A light. The Co(II) complex 1 shows a significant photocytotoxic effect in HeLa cervical cancer cells on exposure to UV-A light of 365 nm, giving an IC(50) value of 32 microM. The IC(50) value for the py-nap ligand alone is 41.42 microM in UV-A light. The IC(50) value is >200 microM in the dark, indicating poor dark toxicity of 1. The Cu(II) complex 2 exhibits moderate photocytotoxicity and significant dark toxicity, giving IC(50) values of 18.6 microM and 29.7 microM in UV-A light and in the dark, respectively.

Two‐Color Two‐Photon Excitation of Intrinsic Protein Fluorescence: Label‐Free Observation of Proteolytic Digestion of Bovine Serum Albumin

Two-color two-photon (2c2p) excitation fluorescence is used to monitor the enzymatic cleavage of bovine serum albumin (BSA) by subtilisin. Fluorescence is generated by irradiation with spatially and temporally overlapping femtosecond laser beams resulting in simultaneous absorption of an 800 and a 400 nm photon. Thereby, excitation of the fluorescent amino acid tryptophan present in BSA corresponds to an effective one-photon wavelength of 266 nm. The progress of protein cleavage is monitored by time-resolved fluorescence analysis. The fluorescence lifetime of tryptophan decreases during the reaction. This demonstrates a novel label-free multiphoton observation technique for conformational changes of proteins containing tryptophan. Due to the strong 2c2p fluorescence signal it is suitable for fast evaluation and monitoring of protein reactions. The course of the reaction is monitored simultaneously by gel electrophoresis. In contrast to conventional one-photon techniques, 2c2p excitation enables label-free protein fluorescence studies without irradiating the sample with UV light. Due to the dependence of the excitation on the power of both laser beams, excitation is limited to a relatively small focal volume. This results in dramatically reduced overall photodamage compared to direct UV irradiation. This method can be easily extended to microscopy imaging techniques.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Conjugated Ternary Copper(II) Complexes

Ferrocene-conjugated ternary copper(II) complexes [Cu(L)(B)](ClO4)2, where L is FcCH2N(CH2Py)2 (Fc = (η5-C5H4)FeII(η5-C5H5)) and B is a phenanthroline base, viz., 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq, 3), and dipyrido[3,2-a:2′,3′-c]phenazine (dppz, 4), have been synthesized and characterized by various spectroscopic and analytical techniques. The bpy complex 1, as its hexafluorophosphate salt, has been structurally characterized by X-ray crystallography. The molecular structure shows the copper(II) center having an essentially square-pyramidal coordination geometry in which L with a pendant ferrocenyl (Fc) moiety and bpy show respective tridentate and bidentate modes of binding to the metal center. The complexes are redox active, showing a reversible cyclic voltammetric response of the Fc+−Fc couple near 0.5 V vs SCE and a quasi-reversible Cu(II)−Cu(I) couple near 0.0 V. Complexes 2−4 show binding affinity to calf thymus (CT) DNA, giving binding constant (Kb) values in the range of 4.2 × 104 to 2.5 × 105 M−1. Thermal denaturation and viscometric titration data suggest groove binding and/or a partial intercalative mode of binding of the complexes to CT DNA. The complexes show good binding propensity to the bovine serum albumin (BSA) protein, giving KBSA values of ∼104 M−1 for the bpy and phen complexes and ∼105 M−1 for the dpq and dppz complexes. Complexes 2−4 exhibit efficient chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent or hydrogen peroxide (H2O2) as an oxidizing agent. Mechanistic studies reveal formation of hydroxyl radicals as the reactive species. The dpq and dppz complexes are active in cleaving supercoiled (SC) pUC19 DNA on photoexposure to visible light of different wavelengths including red light using an argon−krypton mixed gas ion laser. Mechanistic investigations using various inhibitors reveal the formation of hydroxyl radicals in the DNA photocleavage reactions. The dppz complex 4, which shows efficient photoinduced BSA cleavage activity, is a potent multifunctional model nuclease and protease in the chemistry of photodynamic therapy (PDT) of cancer.

Photo-induced double-strand DNA and site-specific protein cleavage activity of l-histidine (μ-oxo)diiron(iii) complexes of heterocyclic bases

Three oxo-bridged diiron(III) complexes of L-histidine and heterocyclic bases [Fe(2)(micro-O)(L-his)(2)(B)(2)](ClO(4))(2) (1-3), where B is 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), were prepared and characterized. The bpy complex was structurally characterized by X- ray crystallography. The molecular structure showed a {Fe(2)(micro-O)} core in which iron(III) in a FeN(4)O(2) coordination is bound to tridentate monoanionic L-histidine and bidentate bpy ligands. The FeFe distance is approximately 3.5 A. The Fe-O-Fe unit is essentially linear, giving a bond angle of approximately 172 degrees . The complexes showed irreversible cyclic voltammetric cathodic response near -0.1 V vs. SCE in H(2)O-0.1 M KCl. The binuclear units displayed antiferromagnetic interaction between two high-spin (S = 5/2) iron(III) centers giving a -J value of approximately 110 cm(-1). The complexes showed good DNA binding propensity giving a binding constant value of approximately 10(5) M(-1). Isothermal titration calorimetric data indicated single binding mode to the DNA. The binding was found to be driven by negative free energy change and enthalpy. The dpq complex showed oxidative double-strand DNA cleavage on exposure to UV-A and visible light. The phen complex displayed single-strand photocleavage of DNA. The DNA double-strand breaks were rationalized from theoretical molecular docking calculations. Mechanistic investigations showed formation of hydroxyl radicals as the reactive species through photodecarboxylation of the L-histidine ligand. The complexes exhibited good binding propensity to bovine serum albumin (BSA) protein in Tris-HCl/NaCl buffer medium. The dpq complex showed UV-A light-induced site-specific oxidative BSA cleavage forming fragments of approximately 45 kDa and approximately 20 kDa molecular weights via OH pathway.

Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis

We characterize here a cDNA from the ixodid tick Haemaphysalis longicornis, which encodes an asparaginyl endopeptidase, legumain (HlLgm), that was present as a functional molecule in the midgut of this tick. Endogenous HlLgm was detected as a 38-kDa antigen in H. longicornis extracts and was seen throughout all developmental stages. Endogenous HlLgm was mainly localized in the midgut epithelium by immunohistochemistry, and was shown to be up-regulated by the host blood-feeding process. Recombinant HlLgm (rHlLgm) produced in Escherichia coli was shown to hydrolyze the synthetic substrate Z-Ala-Ala-Asn-MCA at the rate of 6.42×10 −4 μmol/min/mg protein. Its activity was inhibited by the thiol blocking reagents iodoacetamide and N-ethylmaleimide. The enzyme was shown to possess a unique feature of having an autocatalyzed cleavage at asparagines 364−365 at the C-terminus of both endogenous HlLgm and rHlLgm. rHlLgm degraded bovine hemoglobin and bovine serum albumin (BSA) showing its strict specificity for hydrolysis of the peptide on the carboxyl side of the asparagines, as demonstrated by internal amino acid sequence analysis of proteolytic product of BSA cleavage. These results suggest that HlLgm plays an important role in host blood-meal digestion and may be critical for the final process of digestion of blood components.

Efficient Photocleavage of Lysozyme by a New Chiral Probe

Photocleavage of lysozyme and bovine serum albumin (BSA) by L-phenylalanine-1(1- pyrene)methylamide (PMA-L-Phe) is reported here. The chiral probe, PMA-L-Phe, has a positively charged side chain, while the previous probes carried a free carboxyl group. The yield of lysozyme cleavage by PMA-LPhe is increased to 57% when compared to the previous probes, while the yield of BSA cleavage is reduced to < 5%. Sequencing studies indicated that PMA-L-Phe cleaves lysozyme at a single site, between residues Trp108-Val109. Absorption and fluorescence spectral data indicate that PMA-L-Phe binds to lysozyme and BSA with affinity constants (Kb) of 3.3x105 M-1 and 3.8x105 M-1, respectively. Keywords: pyrene, lysozyme, bovine serum albumin, photocleavage

Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by chromatographic and electrophoretic methods

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by oxo-compounds, particularly glucose, ribose, glyoxal and glutardialdehyde, have been investigated using a set of modern chromatographic and electrophoretic separation methods. High-performance liquid chromatography (HPLC) alternatively with UV spectrophotometric (diode array) or mass spectrometric (MS) detection, polyacrylamide gel electrophoresis (PAGE) with Coomassie brilliant blue staining detection, and capillary zone electrophoresis (CZE) with UV spectrophotometric detection have been employed for the investigation of the chemical and structural changes of BSA caused by its reaction with the above oxo-compounds exhibiting different degree of reactivity. The extent of modifications was found to be dependent on the nature of the oxo-compound used and progressed in the glucose < ribose < glyoxal < glutaraldehyde order. With the aid of HPLC/UV/MS and CZE/UV tryptic peptide mapping and amino acid analysis of both unmodified and modified BSA it was revealed that the mildest modification resulted from the reaction of BSA with glucose, in this case presumably only monofunctional derivatives have arisen, whereas the most intensive modifications were found after BSA reaction with glutardialdehyde, which resulted in high degree of both inter- and intra-molecular cross-linking, due to which no unmodified peptides were detected after tryptic cleavage of BSA modified by this agent.

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease

A chromium(III) complex, transdiaqua [ N , N ′ -propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H 2 O) 2 ]ClO 4 ) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H 2 O) 2 ] + adduct showed a band at λ ex max =370 nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease

A chromium(III) complex, transdiaqua [ N, N ′-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H 2O) 2]ClO 4) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H 2O) 2] + adduct showed a band at λ ex max=370 nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.