Ultraviolet Circular Dichroism Spectra Research Articles

Acid induced gelation of heated soymilks: Protein structure, molecular interactions and gel properties

Heating conditions of soymilks have tremendous effects on the gelation and gel properties of glucono-delta-lactone induced tofu. The defatted soymilk was heated for 5 min at 65–115 °C in a controlled way. Protein structure of heated soymilk were characterized by non-reducing electrophoresis, far ultraviolet circular dichroism and ANS fluorescence spectrum and it was found that surface hydrophobicity and the maximum structural shift was occurred at the temperature of 90 °C. An isothermal titration calorimetry (ITC) based method for the evaluation of protein interactions was developed. Upon acidifying of the soymilks, the experimentally determined exothermic effects were observed but were smaller than the calculated protonation heat. An endothermic effect was proposed to compensate the difference which was most probably resulted from the hydrophobic interactions. The magnitude of the endothermic effect which reflected the strength of the hydrophobic interactions, exhibiting the similar temperature dependence as that of surface hydrophobicity. Gelling behavior and gel properties of the acidified soymilks were studied by rheometer and confocal laser scanning microscopy. The soymilk heated at 90 °C showed the shortest gelation time, the highest storage modulus and the densest gel networks. This study will further deepen the understanding of tofu processing mechanism.

Sinapicacid Inhibits Group IIA Secretory Phospholipase A2 and Its Inflammatory Response in Mice.

Human Group IIA secreted phospholipase A2 (sPLA2-IIA) enzyme plays a crucial role in several chronic inflammatory diseases such asasthma, atherosclerosis, gout, bronchitis, etc. Several studies showed that the antioxidants exert an anti-inflammatory function by inhibiting the sPLA2-IIA enzyme. Hence, the present study evaluated an antioxidant molecule, sinapic acid, for sPLA2-IIA inhibition as an anti-inflammatory function. Initially, the antioxidant efficacy of sinapic acid was evaluated, and it showed greater antioxidant potency. Further, sinapic acid inhibited 94.4 ± 4.83% of sPLA2-IIA activity with an IC50 value of 4.16 ± 0.13 µM. The mode of sPLA2-IIA inhibition was examined by increasing the substrate concentration from 30 to 120nM and the calcium concentration from 2.5 to 15 mM, which did not change the level of inhibition. Further, sinapic acid altered the intrinsic fluorescence and distorted the far UltraViolet Circular Dichroism (UV-CD) spectra of the sPLA2-IIA, indicating the direct enzyme-inhibitor interaction. Sinapic acid reduced the sPLA2-IIA mediated hemolytic activity from 94 ± 2.19% to 12.35 ± 2.57% and mouse paw edema from 171.75 ± 2.2% to 114.8 ± 1.98%, demonstrating the anti-inflammatory efficiency of sinapic acid by in situ and in vivo methods, respectively. Finally, sinapic acid reduced the hemorrhagic effect of Vipera russelli venom hemorrhagic complex-I (VR-HC-I) as an anti-hemorrhagic function. Thus, the above experimental results revealed the sinapic acid potency to be an antioxidant, anti-inflammatory and anti-hemorrhagic molecule, and therefore, it appears to be a promising therapeutic agent.

Structural Analysis of Nonapeptides Derived from Elastin

Elastin-derived peptides are released from the extracellular matrix remodeling by numerous proteases and seem to regulate many biological processes, notably cancer progression. The canonical elastin peptide is VGVAPG, which harbors the XGXXPG consensus pattern, allowing interaction with the elastin receptor complex located at the surface of cells. Besides these elastokines, another class of peptides has been identified. This group of bioactive elastin peptides presents the XGXPGXGXG consensus sequence, but the reason for their bioactivity remains unexplained. To better understand their nature and structure-function relationships, herein we searched the current databases for this nonapeptide motif and observed that the XGXPGXGXG elastin peptides define a specific group of tandemly repeated patterns. Further, we focused on four tandemly repeated human elastin nonapeptides, i.e., AGIPGLGVG, VGVPGLGVG, AGVPGLGVG, and AGVPGFGAG. These peptides were analyzed by means of optical spectroscopies and molecular dynamics. Ultraviolet-circular dichroism and Raman spectra are consistent with a mixture of β-turn, β-strand, and random-chain secondary elements in aqueous media. Quantitative analysis of their conformations suggested that turns corresponded to half of the total population of structural elements, whereas the remaining half were equally distributed between β-strand and unordered chains. These distributions were confirmed by molecular dynamics simulations. Altogether, our data suggest that these highly dynamic peptides harbor a type II β-turn located in their central part. We hypothesize that this structural element could explain their specific bioactivity.

Structural Characterization of the S-glycosylated Bacteriocin ASM1 from Lactobacillus plantarum

In order to protect their environmental niche, most bacteria secret antimicrobial substances designed to target specific bacterial strains that are often closely related to the producer strain. Bacteriocins, small, ribosomally synthesised antimicrobial peptides, comprise a class of such substances and can either inhibit (bacteriostatic) or kill (bactericidal) target cells. Glycocins are a class of bacteriocin that are post-translationally modified by one or more carbohydrate moieties that are either β-O-linked to either a serine or threonine and/or β-S-linked to a cysteine. The solution nuclear magnetic resonance structure (NMR) of the glycocin ASM1 (produced by Lactobacillus plantarum A-1), an orthologue of GccF, has been determined. In both structures, the disulfide bonds are essential for activity and restrict the mobility of the N-acetyl-glucosamine (GlcNAc) attached to Ser-18 (O-linked), compared to the much more flexible GlcNAc moiety on Cys-43 (S-linked). Interestingly, despite 88% sequence identity, the helical structure of ASM1 is less pronounced which appears to be consistent with the far ultra-violet circular dichroism (UV CD) spectra.

Expression, purification, and characterization of pyruvate kinase from Mycobacterium tuberculosis: A key allosteric regulatory enzyme.

The current research aims to isolate pyruvate kinase (Pyk) gene from Mycobacterium tuberculosis and expression of the gene (Rv1617) to obtain a purified enzyme. The enzyme activity and secondary structural features were assessed through biochemical assays and circular dichroism (CD) spectroscopy, respectively. The Pyk-encoding gene from the complete genome of M. tuberculosis was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). The enzyme was purified by nickel-nitrilotriacetic acid affinity chromatography and enzyme activity was determined by a lactose dehydrogenase-coupled assay system. Further, far ultraviolet CD spectra of the enzyme and the substrate bound enzyme were analyzed using a Jasco J712 spectrophotometer. A single protein with an approximate molecular mass of 54 kDa was purified and a specific activity of 5.31 units/mg was determined from purified M. tuberculosis Pyk. The activity of the enzyme indicating a protein is defined by separate domain for each catalytic function. The secondary structure analysis of CD spectra of the recombinant Pyk has revealed a content of 17% α-helix, 34% β-sheet, and 49% turns in the enzyme. The growing evidence has impacted M. tuberculosis central carbon metabolism as a key determinant of the survival and pathogenicity in the host. The purified Pyk was observed to have increased enzyme activity in all steps of purification. Retention of Pyk activity indicates a possible catalytic role for the lower part of the glycolytic pathway. The overall results of the spectra obtained from the CD suggest that the substrate phosphoenolpyruvate and adenosine diphosphate binding to the enzyme can cause conformational changes resulting in the exposure or shielding of residues susceptible to modification.

Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy

Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654cm−1) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402cm−1). For the first time, the correlation between the degree of hydrolysis and the concentration of carboxylic groups measured by FTIR spectroscopy was revealed as well. The far UV-CD spectra together with their secondary structure analysis suggest that the α-helical content decreases concomitant with an increase in the unordered structure. Both spectroscopic techniques also demonstrate that there are similar but less spectral changes of BSA for the trypsin attack than for α-chymotrypsin although the substrate/enzyme ratio is taken the same.

The Role of Calcium in the Conformational Changes of the Recombinant S100A8/S100A9

Calprotectin is a member of the EF-hand proteins, composed of two subunits, S100A8 (MRP8) and S100A9 (MRP14). These proteins are involved in important processes including cell signaling, regulation of inflammatory responses, cell cycle control, differentiation, regulation of ion channel activity and defense against microbial agents in a calcium dependent manner. In the present study, recombinant S100A8 and S100A9 were expressed in E. coli BL21 and then purified using Ni-NTA affinity chromatography. The structure of the S100A8/A9 complex in the presence and absence of calcium was assessed by circular dichroism and fluorescence spectroscopy. The intrinsic fluorescence emission spectra of the S100A8/A9 complex in the presence of calcium showed a reduction in fluorescence intensity, reflecting conformational changes within the protein with the exposure of aromatic residues to the protein surface. The far ultraviolet-circular dichroism spectra of the complex in the presence of calcium revealed minor changes in the regular secondary structure of the complex. Also, increased thermal stability of the S100A8/A9 complex in the presence of calcium was indicated.

Computational Study of the Structure and Electronic Circular Dichroism Spectroscopy of Blue Copper Proteins

The calculation of the electronic circular dichroism (CD) spectra of the oxidized form of the blue copper proteins plastocyanin and cucumber basic protein and the relationship between the observed spectral features and the structure of the active site of the protein is investigated. Excitation energies and transition strengths are computed using multireference configuration interaction, and it is shown that computed spectra based on coordinates from the crystal structure or a single structure optimized in quantum mechanics/molecular mechanics (QM/MM) or ligand field molecular mechanics (LFMM) are qualitatively incorrect. In particular, the rotational strength of the ligand to metal charge transfer band is predicted to be too small or have the incorrect sign. By considering calculations on active site models with modified structures, it is shown that the intensity of this band is sensitive to the nonplanarity of the histidine and cysteine ligands coordinated to copper. Calculation of the ultraviolet absorption and CD spectra based upon averaging over many structures drawn from a LFMM molecular dynamics simulation are in good agreement with experiment, and superior to analogous calculations based upon structures from a classical molecular dynamics simulation. This provides evidence that the LFMM force field provides an accurate description of the molecular dynamics of these proteins.

The (Not Completely Irreversible) Population of a Misfolded State of Cytochrome c under Folding Conditions

This paper reports the discovery of a (meta)stable partially unfolded state of horse heart ferricytochrome c that was obtained after exposing the protein to a solution with an alkaline pH of 11.5 for 1 week. Thereafter, the protein did not undergo any detectable change in its secondary and tertiary structure upon adjusting the solution to folding promoting conditions at neutral pH. Spectroscopic data suggest that the misfolded protein exhibits a hexacoordinated low-spin state with a hydroxyl ion as the likely ligand. Below pH 6, a new ligation state emerges with the spectroscopic characteristics of a pentacoordinated quantum mixed state of the heme iron. Gel electrophoresis revealed substantial formation of soluble dimers and trimers at submillimolar concentrations, whereas monomers were dominant at lower, micromolar concentrations. Ultraviolet circular dichroism spectra indicate that oxidized monomers are pre-molten globule to globule-like with a substantial fraction of secondary (helical) structure reminiscent of alkaline state V. The oligomers contain even more helical structure, which suggests domain swapping as the underlying mechanism of their formation. A substantial fraction of the submillimolar mixture of monomers and oligomers underwent a reduction of the heme iron. Its dependence on pH suggests the coupling to a proton transfer process. Altogether, our data indicate a partially unfolded ferricytochrome c conformation with spectroscopic characteristics reminiscent of the recently discovered alkaline isomer V(b), which is stabilized under folding conditions by exposing the protein to a very alkaline pH for an extended period of time.

Ultraviolet spectroscopy of protein backbone transitions in aqueous solution: combined QM and MM simulations.

A generalized approach combining quantum mechanics (QM) and molecular mechanics (MM) calculations is developed to simulate the n --> pi* and pi --> pi* backbone transitions of proteins in aqueous solution. These transitions, which occur in the ultraviolet (UV) at 180-220 nm, provide a sensitive probe for secondary structures. The excitation Hamiltonian is constructed using high-level electronic structure calculations of N-methylacetamide (NMA). Its electrostatic fluctuations are modeled using a new algorithm, EHEF, which combines a molecular dynamics (MD) trajectory obtained with a MM forcefield and electronic structures of sampled MD snapshots calculated by QM. The lineshapes and excitation splittings induced by the electrostatic environment in the experimental UV linear absorption (LA) and circular dichroism (CD) spectra of several proteins in aqueous solution are reproduced by our calculations. The distinct CD features of alpha-helix and beta-sheet protein structures are observed in the simulations and can be assigned to different backbone geometries. The fine structure of the UV spectra is accurately characterized and enables us to identify signatures of secondary structures.

A significant role for high‐energy transitions in the ultraviolet circular dichroism spectra of polypeptides and proteins

The nπ* and ππ* transitions of polypeptides mix significantly with very high energy transitions in the poly(Pro)II conformation, as evidenced by the strongly nonconservative CD spectrum in the 170-250 nm region. Because of this, the exciton model, the standard quantum mechanical model for predicting absorption and CD spectra of polypeptides, gives poor results for the poly(Pro) II (P(II)) conformation, although it works well for the α-helix, β-sheet, and β-turns. The exciton theory has been extended to include the effects of mixing of discrete peptide transitions near 200 nm with the large number of uncharacterized transitions in the deep ultraviolet. These latter transitions dominate the polarizability, and their mixing with the discrete transitions can be described via bond and lone-pair polarizability tensors, derivable by ab initio methods. This extended exciton method gives a good description of the CD spectrum of (Ala)(n) oligomers in the P(II) conformation. For this conformation, the polarizability contributions lead to a strong negative band near 200 nm that dominates the calculated and observed CD spectrum. The model does not give a good description of the CD of (Pro)(n) oligomers, probably because of conformational heterogeneity or nonadditive contributions of the Pro side chains. The model improves the calculated CD spectra of α-helical (Ala)(n) oligomers. Although the high-energy transitions make only a small net contribution to the CD of the α-helix in the 200 nm region, they enhance the negative exciton band at ∼205 nm and largely cancel the negative exciton band near 175 nm, substantially improving agreement with experiment.

Genistein, a potent inhibitor of secretory phospholipase A2: a new insight in down regulation of inflammation

Some of the legumes, spices and medicinal herbs rich in genistein are known for their anti-inflammatory properties. Anti-inflammatory property of these herbs is determined by subjecting secretory phospholipase A(2) (sPLA(2)) inhibition, a key enzyme in the inflammatory reactions by genistein. Genistein was assessed for inhibition of sPLA(2) activity using (14)C-oleate radiolabelled Escherichia coli membrane as substrate. The enzyme-inhibitor interaction was established by intrinsic fluorescence and circular dichroism studies. The in vivo anti-inflammatory activity was tested by injecting sPLA(2), Vipera russelli venom phospholipase-V (VRV-PL-V) with different concentrations of genistein in the range of 3-21 muM into intra plantar surface of right hind footpad of mice. Systemic effect was tested by administering the genistein (21 muM) i.p. 30 min before and immediately after sPLA(2) injection. Genistein inhibited sPLA(2) enzymes of inflammatory exudates (human synovial fluid and human pleural fluid) and snake venoms (VRV-PL-V and Naja naja phospholipase-I) in a concentration dependent manner with IC(50) values ranging from 5.75 to 11.75 muM. Increasing the calcium (Ca(2+)) concentration from 2.5 to 15 mM and substrate concentration up to 120 nM did not alter the level of inhibition. Genistein alters the intrinsic fluorescence intensity and shown apparent shift in far ultra violet-circular dichroism spectra of VRV-PL-V, indicating the direct interaction with enzyme. Genistein also inhibited the VRV-PL-V induced mouse paw oedema in a concentration dependent manner. The genistein at 21 muM concentration administered immediately after the VRV-PL-V injection, effectively neutralized the oedema inducing activity. Genistein inhibited sPLA(2) activity of both inflammatory exudates and snake venoms in a concentration dependent manner and sPLA(2) induced mouse paw oedema. The study partially explains the observed anti-inflammatory property of several medicinal herbs which containing genistein.

Refolding and purification of the human secreted group IID phospholipase A 2 expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A 2 (sPLA 2s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A 2 (hsPLA 2-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA 2-IID in Escherichia coli, the DNA-coding sequence for hsPLA 2-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA 2-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A 2. The refolded recombinant hsPLA 2-IID demonstrated Ca 2+-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA 2-IID which will advance our understanding of the structure–function relationship and biological effects of the protein.

The potential for circular dichroism as an additional facile and sensitive method of monitoring low-molecular-weight heparins and heparinoids

The ultraviolet circular dichroism (CD) spectra of commercial low-molecular-weight heparins, heparinoids and other anticoagulant preparations have been recorded between 180 and 260 nm. Principal component analysis of the spectra allowed their differentiation into a number of groups related to the means of their production reflecting the structural changes introduced by each process. The findings suggest that CD provides a complementary technique for the rapid analysis of heparin preparations.

Accurate and quick calibration method for polarization-modulation spectroscopy using an ac-modulated polarizing undulator

An accurate calibration method in which an ac-modulated polarizing undulator is used for polarization modulation spectroscopy such as circular dichroism (CD) and linear dichroism (LD) has been proposed and successfully applied to vacuum ultraviolet (vuv) CD and LD spectra measured at beamline BL-5B in the electron storage ring, TERAS, at AIST. This calibration method employs an undulator-modulation spectroscopic method with a multireflection polarimeter, and it uses electronic and optical elements identical to those used for the CD and LD measurements. This method regards the polarimeter as a standard sample for the CD and LD measurements in the vuv region in which a standard sample has not yet been established. The calibration factors for the CD and LD spectra are obtained over a wide range of wavelengths, from 120 to 230 nm, at TERAS BL-5B. The calibrated CD and LD spectra measured at TERAS exhibit good agreement with the standard spectra for wavelengths greater than 170 nm; the mean differences between the standard and calibrated CD and LD spectra are approximately 7% and 4%, respectively. This method enables a remarkable reduction in the experimental time, from approximately 1 h to less than 10 min that is sufficient to observe the storage-ring current dependence of the calibration factors. This method can be applied to the calibration of vuv-CD spectra measured using a conventional photoelastic modulator and for performing an accurate analysis of protein secondary structures.

Human Calprotectin: Effect of Calcium and Zinc on its Secondary and Tertiary Structures, and Role of pH in its Thermal Stability

Calprotectin, a heterodimeric complex belonging to the S100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 (physiological pH) and 8.0 (similar to intestinal pH) was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose-dependent manner. The intrinsic fluorescence emission spectra of human calprotectin (50 microg/ml) in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the alpha-helical content of the protein and an increase in beta- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation.

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.

Calcium-induced conformational changes of the recombinant CBP3 protein from Dictyostelium discoideum

Calcium-binding proteins play various and significant roles in biological systems. Conformational changes in their structures are closely related to their physiological functions. To understand the role of calcium-binding protein 3 (CBP3) in Dictyostelium discoideum, its recombinant proteins were analyzed using circular dichroism (CD) and fluorescence spectroscopy. Gel mobility shift analysis showed that Ca 2+ induced a mobility shift of the recombinant CBP3. Far ultra-violet CD spectra and intrinsic fluorescence spectra on CBP3 and its N- and C-terminal domains exhibited that they underwent a conformational rearrangement depending upon Ca 2+ binding. Measurement of Ca 2+ dissociation constants demonstrated that CBP3 had high affinity toward Ca 2+ in the sub-micromolar range and N-terminal domain had higher affinity than C-terminal domain. The changes of fluorescence spectra by an addition of 8-anilino-1-naphthalene sulfonic acid indicated that the hydrophobic patches of CBP3 and its C-terminal domain are likely to be more exposed in the presence of Ca 2+. Since the exposure of hydrophobic patches is thermodynamically unfavorable, Ca 2+-bound CBP3 may interact with other proteins in vivo. All these data suggest that Ca 2+ induces CBP3 to be more favorable conformation to interact with target proteins.

Shifts in protein charge state distributions with varying redox reagents in nanoelectrospray triple quadrupole mass spectrometry

The influence of a number of redox reagents on the charge state distribution in nanoelectrospray mass spectrometry was examined using cytochrome c and ubiquitin. The redox active species investigated were: 1,4-benzoquinone, quinhydrone, tetracyanoquinodimethane (TCNQ), hydroquinone, and ascorbic acid. The redox active species was mixed with the protein sample before injection into the nanoelectrospray emitter, and mass spectra were acquired using a triple quadrupole mass spectrometer. Under the same experimental conditions, the charge state distribution of cytochrome c was observed to shift from a weighted average charge state of 14.25 (in the absence of redox species) to 7.10 in the presence of 1,4-benzoquinone. When quinhydrone was mixed with cytochrome c, the charge state distribution of the protein also shifted to lower charge states (weighted average charge state = 9.43), indicative of less charge state reduction for quinhydrone than with 1,4-benzoquinone. Addition of the redox reagent had little effect on the conformation of cytochrome c, as indicated by far ultraviolet circular dichroism spectra. In contrast, the reagents TCNQ, hydroquinone, and ascorbic acid exhibited negligible effects on the observed charge state distribution of the protein. The differing results for these redox reagents can be rationalized in terms of the redox half reactions involving these species. The results observed with ubiquitin upon adding quinhydrone were analogous to those observed with cytochrome c.