Polyphosphate amplifies proinflammatory responses of nuclear proteins through interaction with receptor for advanced glycation end products and P2Y1 purinergic receptor.

The extracellular nuclear proteins, histone H4 (H4) and high mobility group box 1 (HMGB1), released by injured cells during the activation of inflammation and coagulation pathways provoke potent inflammatory responses through interaction with pathogen-related pattern recognition receptors (ie, Toll-like receptors [TLRs] and receptor for advanced glycation end products [RAGE]) present on vascular and innate immune cells. Inorganic polyphosphate (polyP) has emerged as a key modulator of coagulation and inflammation. Here, we demonstrate that polyP binds to both H4 and HMGB1 with high affinity, thereby dramatically potentiating their proinflammatory properties in cellular and in vivo models. By using small interfering RNA knockdowns, pharmacologic inhibitors and extracellular domains of the receptors TLR2, TLR4, RAGE, and P2Y1 as competitive inhibitors, we demonstrate that polyP amplifies H4- and HMGB1-mediated inflammatory signaling in human umbilical vein endothelial cells specifically through interaction with the RAGE and P2Y1 receptors, thereby eliciting intracellular Ca(2+) release. Finally, we demonstrate that the natural anticoagulant protease, activated protein C, potently inhibits polyP-mediated proinflammatory effects of both nuclear proteins in cellular and in vivo systems.

Characterization of the protein Z-dependent protease inhibitor interactive-sites of protein Z.

BACKGROUND: Protein Z (PZ) has been reported to promote the inactivation of factor Xa (FXa) by PZ-dependent protease inhibitor (ZPI) by about three orders of magnitude. Previously, we prepared a chimeric PZ in which its C-terminal pseudo-catalytic domain was grafted on FX light-chain (Gla and EGF-like domains) (PZ/FX-LC). Characterization of PZ/FX-LC revealed that the ZPI interactive-site is primarily located within PZ pseudo-catalytic domain. Nevertheless, the cofactor function and apparent Kd of PZ/FX-LC for interaction with ZPI remained impaired ~6-7-fold, suggesting that PZ contains a ZPI interactive-site outside pseudo-catalytic domain. X-ray structural data indicates that Tyr-240 of ZPI interacts with EGF2-domain of PZ. Structural data further suggests that 3 other ZPI surface loops make salt-bridge interactions with PZ pseudo-catalytic domain. To identify ZPI interactive-sites on PZ, we grafted the N-terminal EGF2 subdomain of PZ onto PZ/FX-LC chimera (PZ-EGF2/FX-LC) and also generated two compensatory charge reversal mutants of PZ pseudo-catalytic domain (Glu-244 and Arg-212) and ZPI surface loops (Lys-239 and Asp-293). METHODS: PZ chimeras were expressed in mammalian cells and ZPI derivatives were expressed in Escherichia coli. RESULTS: The PZ EGF2 subdomain fusion restored the defective cofactor function of PZ/FX-LC. The activities of PZ and ZPI mutants were all impaired if assayed individually, but partially restored if the compensatory charge reversal mutants were used in the assay. CONCLUSIONS: PZ EGF2 subdomain constitutes an interactive-site for ZPI. Data with compensatory charge reversal mutants validates structural data that the identified residues are part of interactive-sites. GENERAL SIGNIFICANCE: Insight is provided into mechanisms through which specificity of ZPI-PZ-FXa complex formation is determined.

Bioactive potential of Lagotis cashmeriana: a study on morphology, phytochemicals, and antioxidant activity.

The present study aimed to investigate the morphological features, phytochemicals, phenolic content, and antioxidant activity in different parts of Lagotis cashmeriana. The morphological features depicted that the plant is 7.9 ± 1.699 cm tall with flowers arranged into an inflorescence. The length of inflorescence was 2.597 ± 0.796 cm. Basal leaves were measuring 2.99 ± 0.58 cm. Besides, the number of basal leaves and inflorescence ranged from 4-9 and 0-4 respectively. Methanolic extract of leaves displayed the highest phenolic content (169.5 µg/mL of GAE), followed by inflorescences (157 µg/mL of GAE). Among aqueous extracts, leaves displayed the highest phenolic content (88.38 µg/mL of GAE), followed by inflorescences (76.95 µg/mL of GAE). The results of antioxidant study revealed that the methanolic extracts of leaves possessed the highest antioxidant potential (180.76 µg/mL of AAE). Interestingly, for each extract, there was a positive correlation between the phenolic content and the antioxidant activity.

Role of the residues of the 39-loop in determining the substrate and inhibitor specificity of factor IXa.

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300- to 500-fold. Relative to FXa, AT inhibits FIXa with approximately 40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited approximately 5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with approximately 4-fold decreased k(cat) and approximately 2-fold decreased K(m), although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.

Inhibitory properties of the P1 Tyr variant of antithrombin.

Antithrombin (AT) and protein Z-dependent protease inhibitor (ZPI) are among two physiological serpin inhibitors in plasma that are involved in the regulation of the clotting cascade. Unlike AT, which can inhibit the proteolytic activity of all coagulation proteases, ZPI has narrower protease specificity, inhibiting only factors Xa (fXa) and XIa. Unlike an Arg at the P1 site of the AT reactive center loop (RCL), this residue is a Tyr in ZPI. To investigate the contribution of P1 Tyr in restricting the specificity of ZPI, we engineered an AT mutant in which the P1 Arg of the RCL was replaced with the P1 Tyr of ZPI (AT-R393Y). The reactivity of AT-R393Y with fXa and thrombin was decreased 155- and 970-fold, respectively. However, the serpin mutant inhibited chymotrypsin with an efficiency higher by >4 orders of magnitude. By contrast, chymotrypsin did not exhibit any reactivity with ZPI. The substitution of Asp-189 of fXa with the corresponding residue of chymotrypsin (Ser) did not improve the reactivity of the protease mutant with AT-R393Y; however, the fXa mutant reacted normally with ZPI. These results suggest that the contribution of P1 Tyr to restricting the protease specificity of ZPI is RCL context-dependent and that in addition to P1 Tyr, other structural features within and/or outside the ZPI RCL are involved in determining the protease specificity of the serpin. The results further suggest that thrombin is less tolerant than fXa in accommodating the nonoptimal P1 Tyr of the AT mutant in its active-site pocket.

Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.

Mutagenesis studies toward understanding allostery in thrombin.

The binding of thrombomodulin (TM) to exosite-1 and the binding of Na(+) to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na(+)-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na(+) by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated K(D) to partially restore its activity. Both mutants exhibited approximately 2-3-fold higher K(D) for interaction with Na(+), and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both Na(+)-binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na(+), but TM has no effect on the conformation of this residue.

Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.

Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach (L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 A for APC and 84-86 A for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.

Role of P2 glycine in determining the specificity of antithrombin reaction with coagulation proteases.

Structural data suggests that bulky hydrophobic residues at the S2-S4 sub-sites of factor Xa (fXa) restrict the preference of this pocket for small and non-polar residues like Gly at the P2 position of substrates and inhibitors. However, kinetic studies monitoring the cleavage specificity of 10-residue peptides by fXa have identified Phe as the most preferred P2 residue and Gln-Phe-Arg-Ser-Leu-Ser as the most preferred P3-P3' residues for recognition by fXa. To determine whether this mechanism of specificity is also true for fXa reaction with antithrombin (AT), we prepared two AT mutants having either a Phe at the P2 or Gln-Phe-Arg-Ser-Leu-Ser at the P3-P3' positions of the reactive center loop. Inhibition kinetic studies indicated that the reactivity of P2-Phe with fXa was significantly (approximately 5-fold) impaired, however, the P3-P3' mutant exhibited 1.5-fold improved reactivity with the protease, suggesting cooperative effects between P3-P3' residues influence the P2 specificity of AT. Substitution of Tyr-99 of fXa with a Gly dramatically impaired the reactivity of fXa with wild-type AT, but improved its reactivity with the serpin mutants in the absence, but not in the presence of pentasaccharide. AT with a P2-Phe inhibited thrombin with >150-fold impaired reactivity, however, the defect was restored by either pentasaccharide or by replacing Leu-99 of thrombin with a Gly. The P3-P3' mutant rapidly inhibited factors VIIa and XIa independent of pentasaccharide. These results indicate that P2-Gly plays a key role in determining the S2 sub-site specificity and target protease selectivity of AT in circulation.

Factor Va alters the conformation of the Na+-binding loop of factor Xa in the prothrombinase complex.

Structural and mutagenesis data have indicated that the 220-loop of thrombin is stabilized by a salt-bridge between Glu-217 and Lys-224, thereby facilitating the octahedral coordination of Na (+) with contributions from two carbonyl O atoms of Arg-221a and Lys-224. All three residues are also conserved in fXa and the X-ray crystal structure of fXa indicates that both Glu-217 and Lys-224 are within hydrogen-bonding distance from one another. To investigate the role of these three residues in the catalytic function of fXa and their contribution to interaction with Na (+), we substituted them with Ala and characterized their properties in both amidolytic and proteolytic activity assays. The results indicate that the affinity of all three mutants for interaction with Na (+) has been impaired. The mutant with the greatest loss of affinity for Na (+) (E217A or E217Q) also exhibited a dramatic impairment ( approximately 3-4 orders of magnitude) in its activity toward both synthetic and natural substrates. Interestingly, factor Va (fVa) restored most of the catalytic defect with prothrombin, but not with the synthetic substrate. Both Glu-217 mutants exhibited a near normal affinity for fVa in the prothrombinase assay, but a markedly lower affinity for the cofactor in a direct-binding assay. These results suggest that, similar to thrombin, an ionic interaction between Glu-217 and Lys-224 stabilizes the 220-loop of fXa for binding Na (+). They further support the hypothesis that the Na (+) and fVa-binding sites of fXa are energetically linked and that a cofactor function for fVa in the prothrombinase complex involves inducing a conformational change in the 220-loop of fXa that appears to stabilize this loop in the Na (+)-bound active conformation.

One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism.

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal-dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single-turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of the metallonuclease mechanism. DNA association rate constants are approximately 100-fold higher in the presence of the catalytically nonsupportive Ca(II) versus the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM(2)S and EM(4)S, respectively) support cleavage. Interestingly, the cleavage rate for EM(2)S is approximately 100-fold slower than that displayed by EM(4)S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two-metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.

Autolysis loop restricts the specificity of activated protein C: analysis by FRET and functional assays.

We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S. The activity of APC-Tryp 143-154 toward fVa was identical to that of wild-type APC both in the presence and absence of protein S. However, the reactivity of APC-Tryp 143-154 with plasma inhibitors was preferentially improved independent of protein S. The FRET analysis revealed a dramatic change in the active-site topography of APC both in the absence and presence of protein S. Anisotropy measurements revealed that the fluorescein dye has a remarkable degree of rotational freedom in the active-site of APC-Tryp 143-154. These results suggest that the autolysis loop of APC may not be a target for modulation by protein S. This loop, however, plays a critical role in restricting both the specificity and spatial environment of the active-site groove of APC.

FRET studies with factor X mutants provide insight into the topography of the membrane-bound factor X/Xa.

FRET (fluorescence resonance energy transfer) studies have shown that the vitamin K-dependent coagulation proteases bind to membrane surfaces perpendicularly, positioning their active sites above the membrane surfaces. To investigate whether EGF (epidermal growth factor) domains of these proteases play a spacer function in this model of the membrane interaction, we used FRET to measure the distance between the donor fluorescein dye in the active sites of Fl-FPR (fluorescein-D-Phe-Pro-Arg-chloromethane)-inhibited fXa (activated Factor Xa) and its N-terminal EGF deletion mutant (fXa-desEGF1), and the acceptor OR (octadecylrhodamine) dye incorporated into phospholipid vesicles composed of 80% phosphatidylcholine and 20% phosphatidylserine. The average distance of closest approach (L) between fluorescein in the active site and OR at the vesicle surface was determined to be 56+/-1 A (1 A=0.1 nm) and 63+/-1 A for fXa-desEGF1 compared with 72+/-2 A and 75+/-1 A for fXa, in the absence and presence of fVa (activated Factor V) respectively, assuming kappa2=2/3. In comparison, an L value of 95+/-6 A was obtained for a S195C mutant of fXa in the absence of fVa in which fluorescein was attached directly to Cys(195) of fXa. These results suggest that (i) EGF1 plays a spacer function in holding the active site of fXa above the membrane surface, (ii) the average distance between fluorescein attached to Fl-FPR in the active site of fXa and OR at the vesicle surface may not reflect the actual distance of the active-site residue relative to the membrane surface, and (iii) fVa alters the orientation and/or the height of residue 195 above the membrane surface.

Equilibrium studies of the effect of difference in sequence homology on the mechanism of denaturation of bovine and horse cytochromes-c.

We have carried out equilibrium studies of the effect of the amino acid residue difference in the primary structure of bovine cytochrome-c (b-cyt-c) and horse cyt-c (h-cyt-c) on the mechanism of their folding <--> unfolding processes at pH 6.0 and 25 degrees C. It has been observed that guanidinium chloride (GdmCl)-induced denaturation of b-cyt-c follows a two-state mechanism and that of h-cyt-c is not a two-state process. This conclusion is reached from the coincidence and non-coincidence of GdmCl-induced transition curves of bovine and horse proteins, respectively, monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. These measurements on h-cyt-c in the presence of GdmCl in the concentration range 0.75-2.0 M also suggest that the protein retains all the native far-UV CD but has slightly perturbed tertiary interaction. The intermediate in the presence of these low denaturant concentrations does not have the structural characteristics of a molten globule as judged by the 8-Anilino-1-napthalene sulfonic acid (ANS) binding and near-UV CD experiments. We have also carried out thermal denaturation studies of bovine and horse cyts-c in the presence of GdmCl monitored by absorbance at 405 nm and far-UV CD at 222 nm. The heat-induced denaturation measurements in the presence of the denaturant show (1) that denaturation of b-cyt-c is a two-state process and that of h-cyt-c does not follow a two-state mechanism, and (2) that the enthalpy change on denaturation of both proteins strongly depends on GdmCl concentration.

Contribution of the NH2-terminal EGF-domain of factor IXa to the specificity of intrinsic tenase.

Factor IXa (FIXa) is a vitamin K-dependent coagulation serine protease which binds to factor VIIIa (FVIIIa) on negatively charged phospholipid vesicles (PCPS) to catalyse the activation of factor X (FX) to factor Xa (FXa) in the intrinsic pathway. Fluorescence resonance energy transfer (FRET) studies have indicated that the Gla-domain-dependent interaction of FIXa and FX with PCPS in the presence of FVIIIa positions the active-site of the protease at an appropriate height above the membrane surface to optimise the catalytic reaction. In this study, we investigated the contribution of the NH2-terminal EGF-domain (EGF1) of FIXa to the recognition specificity of intrinsic tenase by constructing an EGF1 deletion mutant of FIXa (FIXa-desEGF1) and characterising the properties of the mutant in kinetic, direct binding and FRET assays. The results of direct binding and kinetic studies demonstrated that the binding affinity of the mutant for interaction with FVIIIa on PCPS has been impaired greater than 10-fold and the catalytic efficiency of the mutant protease-FVIIIa-PCPS complex in the activation of FX has been decreased ~100-fold. By contrast, the mutant protease exhibited a normal activity toward FX in the absence of the protein cofactor. FRET measurements revealed that the distance of the active-site of the mutant FIXa relative to PCPS vesicles has been decreased 10 Å from 75 ± 2 Å for FIXa to 65 ± 2 Å for FIXa-desEGF1 independent of FVIIIa. These results suggest that the NH2-terminal EGF-domain of FIXa provides a binding-site for FVIIIa and plays an essential spacer function in the intrinsic tenase complex.

Protein Z-dependent protease inhibitor binds to the C-terminal domain of protein Z.

Protein Z (PZ) is a multidomain vitamin K-dependent plasma protein that functions as a cofactor to promote the inactivation of factor Xa (fXa) by PZ-dependent protease inhibitor (ZPI) by three orders of magnitude. To understand the mechanism by which PZ improves the reactivity of fXa with ZPI, we expressed wild-type PZ, PZ lacking the gamma-carboxyglutamic acid domain (GD-PZ), and a chimeric PZ mutant in which both Gla and EGF-like domains of the molecule were substituted with identical domains of fXa. The ZPI binding and the cofactor function of the PZ derivatives were characterized in both binding and kinetic assays. The binding assay indicated that all PZ derivatives interact with ZPI with a similar dissociation constant (K(D)) of approximately 7 nm. However, the apparent K(D) for the chimeric PZ-mediated ZPI inhibition of fXa was elevated 6-fold on PC/PS vesicles and its capacity to function as a cofactor to accelerate the ZPI inhibition of fXa was also decreased 6-fold. The cofactor activity of GD-PZ was dramatically impaired; however, the deletion mutant exhibited a normal cofactor function in solution. A chimeric activated protein C mutant containing the Gla domain of fXa was susceptible to inhibition by ZPI in the presence of PZ. These results suggest that: (i) the ZPI interactive site of PZ is located within the C-terminal domain of the cofactor and (ii) a specific interaction between the Gla domains of PZ and fXa contributes approximately 6-fold to the acceleration of the ZPI inhibition of fXa on phospholipid membranes.

Conformational and thermodynamic characterization of the molten globule state occurring during unfolding of cytochromes-c by weak salt denaturants.

The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation <--> X (intermediate) conformation and the second denaturation phase, X conformation <--> D (denatured) conformation are reversible. Conformational characterization of the X state by the far-UV CD, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements led us to conclude that the X state is a molten globule state. Analysis of denaturation transition curves for the stability of different states in terms of Gibbs energy change at pH 6.0 and 25 degrees C led us to conclude that the N state is more stable than the X state by 9.55 +/- 0.32 kcal mol(-1), whereas the X state is more stable than the D state by only 1.40 +/- 0.25 kcal mol(-1). We have also studied the effect of temperature on the equilibria, N conformation <--> X conformation and X conformation <--> D conformation in the presence of different denaturant concentrations using two different optical probes, namely, [theta](222) and Delta epsilon(400). These measurements yielded T(m), (midpoint of denaturation) and Delta H(m) (enthalpy change) at T(m) as a function of denaturant concentration. A plot of Delta H(m) versus corresponding T(m) was used to determine the constant-pressure heat capacity change, Delta C(p) (= ( partial differential Delta H(m)/ partial differential T(m))(p)). Values of Delta C(p) for N conformation <--> X conformation and X conformation <--> D conformation is 0.92 +/- 0.02 kcal mol(-1) K(-1) and 0.41 +/- 0.01 kcal mol(-1) K(-1), respectively. These measurements suggested that about 30% of the hydrophobic groups in the molten globule state are not accessible to the water.