Single disulfide bond reduced papain exists in a compact intermediate state

Partially reduced proteins and other chemically modified derivatives are very useful model systems to understand the protein folding in vivo. Upon reduction, proteins attain different conformations with varying degrees of compactness. The reduction of papain in the presence of 8 M urea leads to the partial reduction of one disulfide bond. This derivative (single disulfide reduced carboxymethylated 1RCM papain (3RCM papain)) was characterized by spectroscopic methods and the effect of this reduction on the unfolding of the protein was investigated. Under this partial reduction, papain exhibits more than half of the tertiary and most of the secondary structures relative to the non-reduced molecule (free cysteine reduced and carboxymethylated papain (1RCM papain)). Hydrophobic regions are exposed to the solvent as observed through 8-anilino-1-naphthalene sulfonic acid binding which was absent in the fully intact and unfolded protein, at neutral pH. Hydrodynamic studies indicated that 3RCM papain, under neutral conditions, possess expanded conformation as compared to the native protein. Tryptophan fluorescence quenching studies suggested the exposure of aromatic residues to solvent. Guanidine hydrochloride induced unfolding of this derivative, at neutral pH, showed a non-cooperative transition contrary to the cooperativity seen with intact protein. Thermal unfolding indicates that 3RCM papain is less stable compared to the intact protein. These findings suggest that partial reduction of papain has a significant effect on the unfolding behavior of papain.     

Preparation and important functional properties of water-soluble chitosan produced through Maillard reaction

The objective of this research was to improve the solubility of chitosan at neutral or basic pH using the Maillard-type reaction method. To prepare the water-soluble chitosans, various chitosans and saccharides were used under various operating conditions. Biological and physicochemical properties of the chitosan-saccharide derivatives were investigated as well. Results indicated that the solubility of modified chitosan is significantly greater than that of native chitosan, and the chitosan-maltose derivative remained soluble when the pH approached 10. Among chitosan-saccharide derivatives, the solubility of chitosan-fructose derivative was highest at 17.1 g/l. Considering yield, solubility and pH stability, the chitosan-glucosamine derivative was deemed the optimal water-soluble derivative. Compared with the acid-soluble chitosan, the chitosan-glucosamine derivative exhibited high chelating capacity for Zn(2+), Fe(2+) and Cu(2+) ions. Relatively high antibacterial activity against Escherichia coli and Staphylococcus aureus was noted for the chitosan-glucosamine derivative as compared with native chitosan. Results suggest that the water-soluble chitosan produced using the Maillard reaction may be a promising commercial substitute for acid-soluble chitosan.     

Refolding of sepharose-bound trypsinogen with disulfide 179 to 203 reduced and carboxymethylated

Disulfide 179 to 203 of native bovine trypsin was reduced with sodium borohydride and converted to the S-carboxymethyl derivative. The modified zymogen was attached to CNBr-activated Sepharose, and the resulting immobilized protein was used in refolding studies. The fully reduced protein was kept at 35°, at pH 8.5, under aerobic conditions, in a mixture of reduced and oxidized glutathione, until the sulfhydryl groups were reoxidized. A maximum yield of 55% was found for the regeneration of S-(carboxymethyl)₂-trypsinogen, and the activated product, S-(carboxymethyl)₂-trypsin, reacted with an active site reagent and gave the expected specific activity toward a typical trypsin substrate. Apparently, the refolding of immobilized S-(carboxymethyl)₂-trypsinogen regenerated the native structure of trypsinogen even though one of the six disulfides could no longer be formed.     

The thermal unfolding of ribonuclease A. A 13C NMR study.

The 13C nuclear magnetic resonance (NMR) spectra of ribonuclease A over the pH range 1-7 and between 6 and 70 degrees C reveal many of the details of its reversible unfolding. Although the unfolding may loosely be described as 'two-state', evidence is presented for intermediate unfolding stages at least 10 degrees C on either side of the main unfolding transition, particularly at low pH. The first residues to unfold are 17-24, in agreement with other results. The C-terminal region shows a steeper temperature dependence of its unfolding than does the main transition, which itself is shown to lead at all pH values to a semi-structured but internally flexible state which is far from being truly random-coil. This is confirmed by measurements of T1 and of nuclear Overhauser enhancement. Indeed, even at pH 1.1 and 70 degrees C there is evidence for considerable motional restriction of cysteine and proline residues, amongst others. The native protein has more variability of structure at low pH than at neutral pH, and also interchanges more rapidly with the semi-structured, denatured state.     

Catalytic activity of Ntau-carboxymethylhistidine-12 ribonuclease: pH dependence.

The pH-dependence of RNAase A and of Ntau-carboxymethylhistidine-12-RNAase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase) catalysis was studied. Apparent acid dissociation constants were obtained by least squares analysis of the kinetics data. These dissociation constants were compared with pKa values of model imidazole compounds, and with pKa values of histidine residues 12 and 119 on the protein. The shapes of the kcat versus pH profiles for RNAase A and its carboxymethyl derivative are very similar, from which it is concluded that the mechanism of catalysis is closely similar in the two proteins. Apparent pKa values obtained from the kinetic data are higher for the carboxymethylated protein than for RNAase A, as are the pKa values of residues 12 and 119. The similar shifts are consistent with the conclusions that both these residues are functionally significant in native and modified enzyme, and that an unblocked tau-nitrogen on histidine-12 is not essential for activity. From the enzyme's catalytic dependence on pH, and the NMR determined pKa values we propose that histidine 12 and 119 function catalytically in their basic and acidic forms respectively.     

Mucoadhesive properties of cross-linked high amylose starch derivatives

Acetate (Ac-), aminoethyl (AE-) and carboxymethyl (CM-)derivatives of cross-linked high amylose starch (HASCL-6) were previously shown to control, over more than 20h, the release of drugs from highly loaded (up to 60% drug) monolithic tablets. It was now of interest to evaluate their mucoadhesive characteristics in view of further utilization in buccal or vaginal transmucosal delivery. The present study shows that ionic AE-HASCL-6 and CM-HASCL-6 derivatives exhibit higher mucoadhesive properties than neutral HASCL-6 and Ac-HASCL-6, suggesting that the ionic groups introduced on cross-linked starch chains play a role in the bioadhesion process. The adhesiveness seemed related to capillary attraction forces. Surface adhesion parameters were calculated for slabs based on the mentioned polymers and corroborated with their swelling behavior at various pH changes. The positively charged AE-derivatives presented a higher adhesion at acidic pH, being thus recommended for vaginal delivery, whereas the negatively charged derivatives (CM-HASCL-6) exhibited a better adhesion at neutral pH, being thus more appropriate for buccal delivery.     

Molecular weight and pH effects of aminoethyl modified chitosan on antibacterial activity in vitro

Aminoethyl modified chitosan derivatives (AEMCSs) with different molecular weight (Mw) were synthesized by grafting aminoethyl group on different molecular weight chitosans and chitooligosaccharide. FTIR, (1)H NMR, (13)C NMR, elemental analysis and potentiometric titration results showed that branched polyethylimine chitosan was synthesized. Clinical Laboratory Standard Institute (CLSI) protocols were used to determine MIC for Gram-negative strain of Escherichia coli under different pH. The antibacterial activity of the derivatives was significantly improved compared with original chitosans, with MIC values against E. coli varying from 4 to 64 μg/mL depending on different Mw and pH. High molecular weight seems to be in favor of stronger antibacterial activity. At pH 7.4, derivatives with Mw above 27 kDa exhibited equivalent antibacterial activity (16 μg/mL), while oligosaccharide chitosan derivative with lower Mw (~1.4 kDa) showed decreased MIC of 64 μg/mL. The effect of pH on antibacterial activity is more complicated. An optimal pH for HAEMCS was found around 6.5 to give MIC as low as 4 μg/mL, while higher or lower pH compromised the activity. Cell integrity assay and SEM images showed evident cell disruption, indicating membrane disruption may be one possible mechanism for antibacterial activity.     

Carbohydrate-containing derivatives of the trypsin-kallikrein inhibitor aprotinin from bovine organs. II. Inhibitor coupled to the (carboxymethyl)dextran derivatives of D-galactose

The trypsin-kallikrein inhibitor aprotinin was coupled to (carboxymethyl)dextran derivatives of D-galactose. The conjugates contained 14 and 38 D-galactose residues/mol of protein, respectively. The apparent dissociation constants Ki of the complexes between trypsin and modified aprotinins proved to be one order of magnitude higher than the respective values for native aprotinin. The distribution of the modified aprotinins in rat organs after endocardial injection has been studied. The conjugates of aprotinin with (carboxymethyl)dextran derivatives of D-galactose were characterized by decreased clearance rates; they accumulated in the active form in liver. The accumulation was 2.5-10 times higher than native aprotinin for the time of observation (5 min-2 h).     

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

Immobilization-stabilization of enzymes; variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multipoint attachment

We have developed a strategy for immobilization-stabilization of trypsin by multipoint covalent attachment to agarose (aldehyde) gels. We have studied the role of four main variables that control the intensity of the trypsin (amine)-agarose (aldehyde) multiinteraction processes: (a) surface density of aldehyde groups in the activated gels, (b) pH of the multiinteraction medium, (c) contact time between insolubilized enzyme and activated support prior to borohydride reduction of the derivatives, and (d) temperature. Different combinations of these four variables have been tested to prepare a number of trypsin-agarose derivatives. All these derivatives preserved 100% of catalytic activity but showed very different stability values. The less stable derivative had exactly the same stability of soluble trypsin in the absence of autolysis phenomena. On the other hand, the three-dimensional structure of the most stable derivative was 5000-fold more stable than the one corresponding to unmodified trypsin. Amino acid analysis of hydrolysates of this very stable derivative reveals that seven lysine residues per trypsin molecule have reacted with the activated support during the process of preparation of the derivative.     

The reactions of phenylglyoxal and related reagents with amino acids.

1. The reaction of phenylglyoxal (PGO), glyoxal (GO), and methylglyoxal (MGO) with amino acids were investigated at mild pH values at 25 degrees. These aldehydes reacted most rapidly with arginine and the rate of reaction increased with increasing pH values. Histidine, cystine, glycine, tryptophan, asparagine, glutamine, and lysine reacted with these aldehydes at significant but various rates, depending on the pH and the kind of the reagent used. The reactions with these amino acids seemed to involve both the alpha-amino groups and the side chain groups, and no significant reaction appeared to occur with the side chain alone except with those of arginine, lysine, and cysteine. These reagents were similarly reactive with the guanidinium group of arginine, but PGO appeared to be much less reactive with the epsilone-amino group of lysine than MGO and GO. The other ordinary amino acids were very much less reactive or did not react at all with these reagents, with the exception of cysteine. 2. Di-PGO-L-arginine was prepared from Nalpha-benzyloxycarbonyl-L-arginine, and di-PGO-methylguanidine from methylguanidine, and the stoichiometry of the reaction of two PGO molecules with one guanidino group was confirmed. A glyoxal derivative of L-arginine (GO-arginine) was prepared by reaction of glyoxal with arginine. GO-arginine was fairly unstable, especially at higher pH values. A similar derivative (MGO-arginine) was also found to be formed by reaction of MGO with L-arginine, and was similarly unstable. These derivatives, however, did not regenerate arginine upon acid hydrolysis.     

Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis.

The parasporal crystalline protoxin of Bacillus thuringiensis contains a single glycoprotein subunit that has a molecular weight of approximately 1.2 X 10(5). The carbohydrate consists of glucose (3.8%) and mannose (1.8%). At alkaline pH, the proendotoxin is apparently solubilized and activated by an autolytic mechanism involving an inherent sulfhydryl protease that renders the protoxin insecticidal. Activation generates protons, degraded polypeptides, sulfhydryl group reactivity, proteolytic activity, and insect toxicity. Chemical modification of the sulfhydryl groups inhibits the proteolytic and insecticidal activities, suggesting that cysteine residues may be present in the active site of the protein.     

Methionine sulfoxide cytochrome c.

Cytochrome c has been chemically modified by methylene blue mediated photooxidation. It is established that the methionine residues of the protein have been specifically converted to methionine sulfoxide residues. No oxidation of any other amino acid residues or the cysteine thioether bridges of the molecule occurs during the photooxidation reaction. The absorbance spectrum of methionine sulfoxide ferricytochrome c at neutrality is similar to that of the unmodified protein except for an increase in the extinction coefficient of the Soret absorbance band and for the complete loss of the ligand sensitive 695 nm absorbance band in the spectrum of the derivative. The protein remains in the low spin configuration which implies the retention of two strong field ligands. Spin state sensitive spectral titrations and model studies of heme peptides indicate that the sixth ligand is definitely not provided by a lysine residue but may be methionine-80 sulfoxide coordinated via its sulfur atom. Circular dichroism spectra indicate that the heme crevice of methionine sulfoxide ferri- and ferrocytochrome c is weakened relative to native cytochrome c. The redox potential of methionine sulfoxide cytochrome c is 184 mV which is markedly diminished from the 260 mV redox potential of native cytochrome c. The modified protein is equivalent to native cytochrome c as a substrate for cytochrome oxidase and is not autoxidizable at neutral pH but is virtually inactive with succinate-cytochrome c reductase. These results indicate that the major role of the methionine-80 in cytochrome c is to preserve a closed hydrophobic heme crevice which is essential for the maintainance of the necessary redox potential.     

Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region

The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12 beta-hydroxy-4-estrene-3,17-dione 12-(bromo[2-14C]acetate) and 3-methoxyestriol 16-(bromo[2-14C]acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12 beta and 16 alpha derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl)cysteine and histidine residues to form either N tau- or N pi-(carboxymethyl)histidine. The inactivated enzyme samples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive N tau- and N phi-(carboxymethyl)histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that the two histidine residues identified in the present study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17 beta-dehydrogenase.     

Effects of pH on conformational properties related to the toxicity of Bacillus thuringiensis delta-endotoxin.

The delta-endotoxin of Bacillus thuringiensis subspecies kurstaki is an intracellular crystalline proteinaceous inclusion which, upon ingestion, is toxic to lepidopteran insects. Upon dissolution at pH > 9 it yields a protein subunit called protoxin. Under appropriate conditions, protoxin is hydrolyzed to a toxin molecule, which is responsible for killing the insect. It is known that this toxic activity decreases considerably above pH 10. In this study, circular dichroism spectroscopy has been used to examine the secondary structures of the protoxin and toxin molecules at different pH values to determine if there are detectable conformational changes associated with their pH-dependent functional properties. At pH 10, where toxic activity is approximately maximal, both the protoxin and toxin molecules were found to assume a conformation that is on an average approx. 26% alpha-helix and approx. 45% beta-structure. As the pH was increased above 10, where the insecticidal activity decreases, the magnitude of the CD spectrum at 222 nm decreased for protoxin and the calculated alpha-helix contents of both protoxin and toxin molecules decreased. The net secondary structure did not change significantly at pH values below 10. Significant conformational differences are observed between the secondary structure of the protoxin and toxin molecules at different pH values. The pH-dependent changes in secondary structure of the protoxin and toxin can be correlated with the effects of pH on the insecticidal activity of these proteins.     

Chemical-proteomic strategies to investigate cysteine posttranslational modifications

The unique combination of nucleophilicity and redox-sensitivity that is characteristic of cysteine residues results in a variety of posttranslational modifications (PTMs), including oxidation, nitrosation, glutathionylation, prenylation, palmitoylation and Michael adducts with lipid-derived electrophiles (LDEs). These PTMs regulate the activity of diverse protein families by modulating the reactivity of cysteine nucleophiles within active sites of enzymes, and governing protein localization between soluble and membrane-bound forms. Many of these modifications are highly labile, sensitive to small changes in the environment, and dynamic, rendering it difficult to detect these modified species within a complex proteome. Several chemical-proteomic platforms have evolved to study these modifications and enable a better understanding of the diversity of proteins that are regulated by cysteine PTMs. These platforms include: (1) chemical probes to selectively tag PTM-modified cysteines; (2) differential labeling platforms that selectively reveal and tag PTM-modified cysteines; (3) lipid, isoprene and LDE derivatives containing bioorthogonal handles; and (4) cysteine-reactivity profiling to identify PTM-induced decreases in cysteine nucleophilicity. Here, we will provide an overview of these existing chemical-proteomic strategies and their effectiveness at identifying PTM-modified cysteine residues within native biological systems.     

A rapid procedure for assaying nicotinamide phosphoribosyltransferase

We examined the potential use of bovine enterokinase for the limited proteolysis of proteins containing sequences of one or more acidic residues preceding a basic residue. Proteolysis was followed by observing the appearance of fragments by sodium dodecyl sulfate-gel electrophoresis. The susceptible peptide bond was identified from a knowledge of the size of the fragment and the amino acid sequence of the protein. Bovine serum albumin was resistant to proteolysis in its native state, was somewhat susceptible as the S-carboxyamidomethyl derivative, and was highly susceptible as the S-carboxymethyl derivative. S-Alkylated soybean trypsin inhibitor and hen egg white lysozyme were both susceptible to limited hydrolysis, but only in the presence of deoxycholate. All susceptible bonds were either lysine or arginine. The preceding acidic residues could be either aspartic acid, glutamic acid, or carboxymethyl cysteine. If a single acidic residue immediately preceded the basic residue, the rate of hydrolysis was slow. The rate of hydrolysis was also slow if a carboxymethyl cysteine was introduced at the position following the basic residue. In addition to better defining the specificity of enterokinase, these results indicate that enterokinase may be useful in amino acid sequence studies for the production of large fragments. The enzyme may also be useful in DNA-recombinant studies in releasing the desired polypeptide chain from neighboring sequences.     

Preparation and some properties of immobilized trypsin from the crayfish Cambarus affinis Say (author's transl)]

The anionic tryptic enzyme from the crayfish (crayfish trypsin) was adsorbed to DEAE-Sephadex A-50 and covalently coupled to BrCN-activated Sepharose 4B and porous glass loaded with isothiocyanate propyl groups (ITC-glass). The relative activities against p-tosylarginine methyl ester (TosArgOMe) were found to be 30 to 100% for DEAE-Sephadex crayfish trypsin, 20 to 53% for Sepharose crayfish trypsin, and 17 to 38% for ITC-glass crayfish trypsin. The relative activities rise with declining protein content of the enzyme matrix complexes. The highest relative proteinase activities (substrate: 1% casein) were obtained with Sepharose crayfish trypsin (74%), followed by DEAE-Sephadex crayfish trypsin (68%) and ITC-glass crayfish trypsin (45%). Similar results are obtained with protamine and native lactate dehydrogenase as substrates. In accordance with the Sepharose bovine trypsin complex the apparent Michaelis constant (Km(app)) of the Sepharose crayfish trypsin with TosArgOMe was found to be markedly higher than that of the native enzyme. The pH-activity profiles of the crayfish trypsin derivatives using TosArgOMe as substrate were shown to be displaced towards more alkaline pH values by 0.5 (ITC-glass crayfish trypsin) and 1 (Sepharose crayfish trypsin) pH units, respectively, or towards more acidic pH values (by 1.5 pH units) with the polycationic derivative (DEAE-Sephadex crayfish trypsin) as compared to the native enzyme (optimum pH 8.6). Concerning the temperature stability of the derivatives, Sepharose crayfish trypsin was more stabile, ITC-glass crayfish trypsin behaves like the native crayfish trypsin, and DEAE-Sephadex crayfish trypsin was more sensitive at elevated temperatures as compared to the soluble enzyme. The properties of the crayfish trypsin derivatives are compared with the properties of the bovine analogues.     

Selective cysteine modification of metal‐free human metallothionein 1a and its isolated domain fragments: Solution structural properties revealed via ESI‐MS

Human metallothionein 1a, a protein with two cysteine‐rich metal‐binding domains (α with 11 Cys and β with 9), was analyzed in its metal‐free form by selective, covalent Cys modification coupled with ESI‐MS. The modification profiles of the isolated β‐ and α‐fragments reacted with p‐benzoquinone (Bq), N‐ethylmalemide (NEM) and iodoacetamide (IAM) were compared with the full length protein using ESI‐mass spectral data to follow the reaction pathway. Under denaturing conditions at low pH, the reaction profile with each modifier followed pathways that resulted in stochastic, Normal distributions of species whose maxima was equal to the mol. eq. of modifier added. Our interpretation of modification at this pH is that reaction with the cysteines is unimpeded when the full protein or those of its isolated domains are denatured. At neutral pH, where the protein is expected to be folded in a more compact structure, there is a difference in the larger Bq and NEM modification, whose reaction profiles indicate a cooperative pattern. The reaction profile with IAM under native conditions follows a similar stochastic distribution as at low pH, suggesting that this modifier is small enough to access the cysteines unimpeded by the compact structure. The data emphasize the utility of residue modification coupled with electrospray ionization mass spectrometry for the study of protein structure.     

Oxidation of methionine residues in coagulation factor VIIa

The oxidation of the activated form of recombinant coagulation factor VII (FVIIa) by hydrogen peroxide has been studied. The three predominant oxidation products observed at pH 7.5 have been characterized as methionine sulfoxide derivatives of the parent protein involving two of the four methionine residues of the protein, Met298 and Met306. We conclude that oxidation of FVIIa with hydrogen peroxide only affects methionine residues and selectively oxidizes those which are readily accessible to the solvent. The oxidation process has been studied in the pH range 3.5-9.5. The total rate of oxidation of FVIIa as well as the formation of the three oxidation products is consistent over the pH interval 7.5-9.5. However, under acidic conditions, significant variations have been observed indicating a conformational change of FVIIa. Oxidized FVIIa had the same amidolytic activity as the native protein. The binding to soluble tissue factor (TF) was weaker after oxidation as manifested by a threefold increase in dissociation constant and the amidolytic activity in complex with soluble TF was 80% compared to that of native FVIIa. In complex with lipid surface TF, the rate of factor X activation catalyzed by oxidized FVIIa was also reduced by approximately 20% compared to that of native FVIIa. However, native and oxidized FVIIa appeared to bind lipidated TF with indistinguishable affinities.