Structural requirements for thymosin beta4 in its contact with actin. An NMR-analysis of thymosin beta4 mutants in solution and correlation with their biological activity.

We examined the conformational preferences of mutants of thymosin beta4, an actin monomer sequestering protein by NMR spectroscopy in 60% (v/v) trifluoroethanol. Under these conditions, the wild-type thymosin beta4 conformation consists of an alpha-helix (helix I) extending from residues 5-16 with a more stable fragment from lysine 11 to lysine 16 and a second alpha-helix (helix II) encompassing residues 31-39. The point mutations studied here are located in helix I or in the LKKTET segment (residues 17-22) that form the two main entities of interaction with the actin molecule. The alpha-1H conformational shifts allow us to investigate the helicity of the polypeptides at the residue level and to correlate these structures with their biological activity. We determine that an extension of helix I at its C-terminal end over the LKK-segment results in loss of activity. The correct termination of this helix is connected to a specific orientation of the polypeptide essential for a cooperative action of the thymosin beta4 binding entities required for full activity.     

Effect of the N-terminal glycine on the secondary structure, orientation, and interaction of the influenza hemagglutinin fusion peptide with lipid bilayers.

The amino-terminal segment of the membrane-anchored subunit of influenza hemagglutinin (HA) plays a crucial role in membrane fusion and, hence, has been termed the fusion peptide. We have studied the secondary structure, orientation, and effects on the bilayer structure of synthetic peptides corresponding to the wild-type and several fusogenic and nonfusogenic mutants with altered N-termini of the influenza HA fusion peptide by fluorescence, circular dichroism, and Fourier transform infrared spectroscopy. All peptides contained segments of alpha-helical and beta-strand conformation. In the wild-type fusion peptide, 40% of all residues were in alpha-secondary and 30% in beta-secondary structures. By comparison, the nonfusogenic peptides exhibited larger beta/alpha secondary structure ratios. The order parameters of the helices and the amide carbonyl groups of the beta-strands of the wild-type fusion peptide were measured separately, based on the infrared dichroism of the respective absorption bands. Order parameters in the range 0.1-0.7 were found for both segments of the wild-type peptide, which indicates that they are most likely aligned at oblique angles to the membrane normal. The nonfusogenic but not the fusogenic peptides induced splitting of the infrared absorption band at 1735 cm(-1), which is assigned to stretching vibrations of the lipid ester carbonyl bond. This splitting, which reports on an alteration of the hydrogen bonds formed between the lipid ester carbonyls and water and/or hydrogen-donating groups of the fusion peptides, correlated with the beta/alpha ratio of the peptides, suggesting that unpaired beta-strands may replace water molecules and hydrogen-bond to the lipid ester carbonyl groups. The profound structural changes induced by single amino acid replacements at the extreme N-terminus of the fusion peptide further suggest that tertiary or quaternary structural interactions may be important when fusion peptides bind to lipid bilayers.     

Conformational features of a hexapeptide model Ac-TGAAKA-NH2 corresponding to a hydrated alpha helical segment from glyceraldehyde 3-phosphate dehydrogenase: implications for the role of turns in helix folding

Recent analysis of alpha helices in protein crystal structures, available in literature, revealed hydrated alpha helical segments in which, water molecule breaks open helix 5-->1 hydrogen bond by inserting itself, hydrogen bonds to both C=O and NH groups of helix hydrogen bond without disrupting the helix hydrogen bond, and hydrogen bonds to either C=O or NH of helix hydrogen bond. These hydrated segments display a variety of turn conformations and are thought to be 'folding intermediates' trapped during folding-unfolding of alpha helices. A role for reverse turns is implicated in the folding of alpha helices. We considered a hexapeptide model Ac-1TGAAKA6-NH2 from glyceraldehyde 3-phosphate dehydrogenase, corresponding to a hydrated helical segment to assess its role in helix folding. The sequence is a site for two 'folding intermediates'. The conformational features of the model peptide have been investigated by 1H 2D NMR techniques and quantum mechanical perturbative configuration interaction over localized orbitals (PCILO) method. Theoretical modeling largely correlates with experimental observations. Based upon the amide proton temperature coefficients, the observed d alpha n(i, i + 1), d alpha n(i, i + 2), dnn(i, i + 1), d beta n(i, i + 1) NOEs and the results from theoretical modeling, we conclude that the residues of the peptide sample alpha helical and neck regions of the Ramachandran phi, psi map with reduced conformational entropy and there is a potential for turn conformations at N and C terminal ends of the peptide. The role of reduced conformational entropy and turn potential in helix formation have been discussed. We conclude that the peptide sequence can serve as a 'folding intermediate' in the helix folding of glyceraldehyde 3-phosphate dehydrogenase.     

Synthesis, conformational analysis, and spectroscopic characterization of peptides based on Daf, the first rigid transition-metal receptor, cyclic C(alpha,alpha)-disubstituted glycine

Three series of terminally protected model oligopeptides to the nonamer level, based on 9-amino-4,5-diazafluorene-9-carboxylic acid, the first rigid bipyridine-type C(alpha,alpha)-disubstituted glycine, and either Gly, L-Ala, or Aib residues were synthesized by solution methods and fully characterized. The molecular structures of two derivatives and one tripeptide were determined in the crystal state by x-ray diffraction. Moreover, the solution preferred conformations of these peptides were assessed by Fourier transform infrared absorption and (1)H-NMR techniques. A comparison with the known structural tendencies of the strictly related C(alpha,alpha)-disubstituted glycyl residues 1-aminocyclopentane-1-carboxylic acid and 9-aminofluorene-9-carboxylic acid is made, and the implications for the use of the 9-amino-4,5-diazafluorene-9-carboxylic acid residue in conformationally constrained analogs of bioactive peptides are briefly examined. A spectroscopic (uv absorption, fluorescence, CD) characterization of this novel heteroaromatic C(alpha,alpha)-disubstituted glycine is also reported. Finally, preliminary conformational data and membrane activity measurements are discussed for an analog of the lipopeptaibol antibiotic [L-Leu(11)-OMe] trichogin GA IV in which a 9-amino-4,5-diazafluorene-9-carboxylic acid residue was synthetically incorporated in position 1 (replacing the original Aib residue).     

The complete chirospectroscopic signature of the peptide 3(10)-helix in aqueous solution

We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution.     

The conformation of peptide thymosin α1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. a possible model for its interaction with the lymphocyte membrane

The 28-residue peptide thymosin α1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin α1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin α1 has a structure characterized by two regions. A β-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an α helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin α1.     

Appearance of thymosin alpha 1 in supernatants of monocytes incubated with prothymosin alpha.

Prothymosin alpha, a polypeptide of 109 to 111 amino acid residues, contains the entire thymosin alpha 1 sequence (residues 1-28) at its amino terminal. Human peripheral blood monocytes incubated with prothymosin alpha release thymosin alpha 1 in the culture supernatants. In addition total RNA is found to increase. The production of thymosin alpha 1 involves de novo protein synthesis as shown by the kinetics of this release and its inhibition by actinomycin D and cycloheximide. Thymosin alpha 1 release, possibly in association with HLA-DR, stimulates the proliferation of the T cell population.     

Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model.

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.     

Chemical synthesis of proteins in solution

Development of a novel strategy suitable for the solution synthesis of proteins is described, wherein the entire molecule is assembled from fully protected segments in the size range of about 10 residues. Each segment is designed so as to have a common structure of Boc-peptide-OPac (Pac: phenacyl) and all of the side-chain functional groups are protected by Bzl-based groups. New types of solvent systems are described for dissolving fully protected segments in which the segment condensation reactions in solution can be carried out smoothly. After removal of the Boc or Pac group, the segments are coupled together to obtain the entire sequence using the 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide/3, 4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine method. The side-chain protecting groups are then removed by HF and the liberated peptide is subjected to folding reactions to obtain the native conformation. Applying the strategy, the 123-residue human angiogenin, the 121-residue human midkine, the 136-residue human pleiotrophin, and the 238-residue Aequoria green fluorescent protein were synthesized successfully.     

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.     

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.     

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of the binding and structure of adrenocorticotropin peptides in membrane mimics by NMR spectroscopy and pulsed-field gradient diffusion.

The partition and structure of three adrenocorticotropic hormone peptides ACTH(1-10), ACTH(1-24), and ACTH(11-24) in water and in sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles were studied by 2D NMR and NMR gradient diffusion measurements. The diffusion rates, the NH chemical shifts, and the nuclear Overhauser effect patterns provided a coherent picture of binding of these peptides. All three peptides are significantly partitioned in the negatively charged SDS micelles and possess definite secondary structure, as opposed to random structures in water. For ACTH (1-24), the hydrophobic 1-10 segment is partitioned in DPC micelles, but the charged 11-24 segment prefers to remain in the aqueous region. ACTH(11-24) does not bind significantly to the DPC micelles. The binding of the ACTH peptides in these two widely used "membrane mimics" are substantially different from that in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers obtained by attenuated total reflection infrared spectroscopy and from our preliminary diffusion studies of the same peptides in POPC vesicles. This study showed that, in a given micellar medium, all corresponding segments of these peptides are located in the same membrane environment in the system, regardless of whether these segments exist by themselves or are attached to other segments. This result may contradict the membrane-compartments concept of Schwyzer, which suggests that ACTH(1-10) and ACTH(1-24) are located in different membrane compartments because they have different address segments, and consequently, bind to different receptors. The present results also suggest that the assumption that micelles are good membrane mimics should be carefully examined.     

Structure and immunological properties of thymosin beta 9 Met, a new analog of thymosin beta 4 isolated from porcine thymus

During the course for the studies of thymosin beta 4 and prothymosin alpha from porcine thymus, a new analog of thymosin beta 4 has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin beta 9 except that leucine at position 6 in beta 9 is substituted by methionine. Thus, this new peptide has been termed thymosin beta 9 Met. The recoveries of beta 9 Met, beta 4, and prothymosin alpha in porcine tissues have been determined (in micrograms/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin beta 9 Met was found to be more active than beta 4 in enhancing gamma-interferon production in cord blood lymphocytes. However, beta 4 appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.     

Fourier transform infrared analysis of bacteriorhodopsin secondary structure.

Fourier transform infrared spectroscopy at a resolution of 1 cm-1 has been used to study the conformation of dark-adapted bacteriorhodopsin in the native purple membrane, in H2O and D2O suspensions. A detailed analysis of the amide I bands was made using derivative and deconvolution techniques. Curve-fitting results of four independent experiments indicate, after estimation of the methodological errors, that native bacteriorhodopsin contains 52-73% alpha-helices, 13-19% reverse turns, 11-16% beta-sheets, and 3-7% unordered segments. Our analysis has enabled the identification of several components corresponding to alpha-helices, beta-sheets, and reverse turns. Besides the alpha I- and alpha II-helices (peaking at 1658 and 1665 cm-1), we propose that two more infrared bands arise from alpha-helical structures: one at 1650 cm-1 from alpha I and another one at 1642 cm-1 in H2O suspension, which could originate from type III beta-turns (i.e., one turn of 3(10)-helix). The relatively high content of reverse turns suggests the presence of one reverse turn per loop, plus another one in the C-terminal segment. On the other hand, several reasons argue that the calculated mean beta-sheet content of around 14% should be decreased somewhat. These beta-sheets could be located in the noncytoplasmatic links of the bacteriorhodopsin molecule.     

Simultaneous isolation and determination of prothymosin alpha, parathymosin alpha, thymosin beta 4, and thymosin beta 10

A method was described for the isolation of peptides from rat thymus. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from rat thymus were, in micrograms per gram of whole tissue, 60-80 for prothymosin alpha, 50-80 for thymosin beta 4, and 20-30 for thymosin beta 10. The procedure also yielded smaller quantities of a fourth peptide, designated parathymosin alpha. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin alpha and thymosin beta 4 to the boiled tissue extract.     

Folding landscapes of the Alzheimer amyloid-beta(12-28) peptide

The energy landscape for folding of the 12-28 fragment of the Alzheimer amyloid beta (Abeta) peptide is characterized using replica-exchange molecular dynamics simulations with an all-atom peptide model and explicit solvent. At physiological temperatures, the peptide exists mostly as a collapsed random coil, populating a small fraction (less than 10%) of hairpins with a beta-turn at position V18F19, with another 10% of hairpin-like conformations possessing a bend rather than a turn in the central VFFA positions. A small fraction of the populated states, approximately 14%, adopt polyproline II (PPII) conformations. Folding of the structured hairpin states proceeds through the assembly of two locally stable segments, VFFAE and EDVGS. The interactions stabilizing these locally folded structural motifs are in conflict with those stabilizing the global fold of A12-28, a signature of underlying residual frustration in this peptide. At increased temperature, the population of both beta-strand and PPII conformations diminishes in favor of beta-turn and random-coil states. On the basis of the conformational preferences of Abeta 12-28 monomers, two models for the molecular structure of amyloid fibrils formed by this peptide are proposed.     

Protected 1-3 segment of the peptaibol antibiotics alamethicin and hypelcin. Solid-state and solution study of a stereochemically constrained linear peptide

A study of the modes of folding and self-association of Z-Aib-L-Pro-Aib-OMe (the protected 1-3 segment of the peptaibol antibiotics alamethicin and hypelcin) in the solid state was performed using i.r. absorption and X-ray diffraction. The stereochemically constrained tripeptide molecules adopt a 4 leads to 1 intramolecularly H-bonded form (beta-turn), where the single intramolecular H-bond is found between the peptide N-H group of the Aib3 residue and the urethane C = O group of the N-blocking benzyloxycarbonyl moiety. This folded structure is stabilized by an intermolecular H-bond between the urethane N-H group of the Aib1 residue and the peptide C = O group of the Pro2 residue of a symmetry related molecule. According to the i.r. absorption data, in CH2Cl2 and TMP solutions the same intramolecularly H-bonded form occurs as that found in solid state. Compared to the situation in the solid state, in CH2Cl2 and TMP solvation of the urethane N-H group replaces self-association (through the same N-H group). The results are also discussed in relation to those obtained for other protected -Aib-X-Aib- (X = Aib, L-Ala, L-Val) tripeptide segments of peptaibol antibiotics.     

Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor]

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.     

Hydrogen-isotope exchange of oxidized and reduced cytochrome c. A comparison of mass spectrometry and infrared methods.

Hydrogen-deuterium exchange in 2H20 solutions of the two redox states of horse heart cytochrome c was investigated at 20 degrees C, pH 7, by mass spectrometry and infrared spectroscopy. Mass spectrometry indicates that ferricytochrome has 20 hydrogens unexchanged after 24 h, 28 hydrogens exchanging between 10 min and 24 h, and 156 hydrogens exchanging within 10 min; comparative values for ferrocytochrome are 45, 19 and 140. The displacement of the exchange curves obtained by infrared corresponds to 8 to 9 peptide hydrogens. These combined methods show many non-peptide hydrogens exchanging rapidly (87 and 79 for ferricytochrome c and ferrocytochrome c respectively), whereas others, probably buried inside the molecule and involved in hydrogen bonds, are not exchanged, even after 24 h (14 and 30 hydrogens respectively, which is relatively large for a small protein). Infrared results are given in terms of changes of standard free energy for the transconformational reaction which exposes the peptide hydrogens to solvent: in ferricytochrome c and ferrycoytochrome c, 30% and 40% respectively of the peptide hydrogens are protected by conformational transitions stabilized by more than 5 kcal/mol (21 kJ/mol), which implies a large increase in rigidity for the reduced form.