Release of intact, monomeric cytochrome c from apoptotic and necrotic cells.

Cytochrome c (Cyt c) has been shown to translocate from mitochondria to the cytoplasm of cells early in apoptosis. In this study sandwich ELISAs for Cyt c were used to determine if Cyt c is ultimately released from apoptotic and necrotic cells. Gel-filtration and cation-exchange chromatographies, in conjunction with immunoreactivity in ELISA, and Western blotting were employed to examine the integrity of the released Cyt c. The results show that Cyt c is released from both apoptotic and necrotic cells in an intact, monomeric form. The release of Cyt c from apoptotic splenocytes began within 2 h following apoptotic insult, while Cyt c was immediately released following induction of necrosis by heat shock. These findings may be relevant to understanding how Cyt c becomes a target for antibody production in some patients with systemic autoimmune diseases.

Cytochrome-C localizes in secretory granules in pancreas and anterior pituitary.

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome-c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome-c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome-c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome-c which recognize an epitope not present in rat cytochrome-c, (ii) preadsorption of antibodies with purified cytochrome-c, and (iii) omission of the primary antibody. The indicated presence of cytochrome-c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome-c.

Immunoglobulin gene joints compensate for reduced on-rates imposed by somatic mutations in a V(H) gene.

Affinity maturation in the B lymphocyte response to a protein epitope appears to be largely due to a decrease in the off-rate constant of the antibodies (Ab) resulting from somatic mutation without a significant increase in the on-rate constant. Here, we show by site-directed mutagenesis of a germline encoded single-chain Fv that somatic mutations frequently selected in the Ab response to mouse cytochrome c (CYT) at heavy (H) chain positions 31 and 58 actually cause a two and three-fold decrease, respectively, in the on-rate constant as well as a two and five-fold decrease, respectively, in the off-rate constant and together cause nearly an eight-fold decrease in the off-rate. However, additional selection for a tyrosine residue at position 96 in the V(kappa)-J(kappa) joint compensates for the decreased on-rate imposed by the somatic mutations. This allows for an increase in the affinity of Ab during the secondary response. Certain sequences at the V(H)-D-J(H) joint were also shown to maintain a normal on-rate constant in the context of the common H chain mutations and, in addition, to reduce the off-rate, thus increasing the affinity. The results support the idea that both faster on-rates and slower off-rates for B lymphocyte antigen-specific receptors are favored during the maturation of the Ab response to mouse CYT.

Antibody-detected folding: kinetics of surface epitope formation are distinct from other folding phases.

The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected (ad) kinetic phase accompanying epitope formation has k(ad) = 0.2 s(-1) and is approximately 40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c (k2f approximately 8 s(-1)), but occurs prior to the absorbance- and fluorescence-detected slow folding steps (k1a approximately 0.06 s(-1); k1b approximately 0.09 s(-1)). N5I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.

Kinetic and genetic bases for the heteroclitic recognition of mouse cytochrome c by mouse anti-pigeon cytochrome c monoclonal antibodies.

The B lymphocyte response to pigeon cytochrome c (CYT) in BALB/c mice was previously shown to initiate as a heteroclitic response specific for the self antigen mouse CYT. As the immune response progressed, the mAb that were produced became less heteroclitic and often bound pigeon CYT with higher affinity than they bound mouse CYT [Minnerath, J. et al., 1995. Proc. Natl. Acad. Sci. USA 92, 12379-12383]. To study the basis for heterocliticity and its loss in this system, the H and L chain amino acid sequences of anti-pigeon CYT mAb obtained from the primary and secondary Ab responses were first compared. The most frequent somatic mutations and Ig gene joints were then introduced into an engineered single-chain Fv (scFv) that expressed the germline-encoded V(H) and V(L) amino acid sequences. The effects of those changes on the on- rate, off-rate, and affinity constants in binding both mouse and pigeon CYT were determined by surface plasmon resonance. In this system, the heterocliticity of primary mAb was largely due to a decreased on-rate constant for binding pigeon CYT relative to mouse CYT. Various combinations of the three frequently occurring H chain somatic mutations (H31, H56, and H58) increased the on-rate constant to maximal levels. Only one of the mutations (H58) decreased the off-rate constant when in combination with the other mutations and the effect of H58 was greater for scFv binding mouse CYT than pigeon CYT. Consequently, the mutated scFv and many secondary mAb remained heteroclitic, although their affinities for pigeon CYT increased. Secondary mAb that were no longer heteroclitic expressed non-canonical amino acid sequences in the V(H)-D-J(H) joint in the context of the canonical V genes or expressed different V genes. In addition to providing insight into the molecular basis for heterocliticity, our findings confirm that both faster on-rate and slower off-rate constants are favored during affinity maturation of the Ab response.

Characterization of monoclonal antibodies to an immunodominant protein of the etiologic agent of human granulocytic ehrlichiosis.

Immunodominant proteins in the range of 42-45 kD are important for the serodiagnosis of human granulocytic ehrlichiosis (HGE). Antigens from human isolates of the etiologic agent of HGE cultivated in HL-60 cells were used to immunize BALB/c mice and generate a panel of hybridomas secreting monoclonal antibodies. Using an enzyme immunoassay, an immunofluorescent assay (IFA), and Western blotting, we showed that culture supernatants and ascites of these hybridomas were reactive with human isolates of the etiologic agent of HGE, Ehrlichia equi and E. phagocytophila. Following screening and subcloning, we selected three stable hybridomas, R1B10, R5E4, and R5A9, which were determined to be of the isotypes IgG3, IgG1, and IgG2a, respectively. These results suggest that the epitopes of the 42-45-kD protein recognized by these three monoclonal antibodies are conserved among E. equi, E. phagocytophila, and the etiologic agent of HGE. Western blot analysis showed reactivity with the 44-kD protein of human isolates of the HGE agent. None of the monoclonal antibodies were reactive with HL-60 cells that were not infected with the HGE agent. No cross-reactivity with related intracellular pathogens could be detected when undiluted supernatants from hybridoma cultures were allowed to react by IFA with antigens from E. chaffeensis, E. risticii, E. platys, Rickettsia rickettsii, R. prowazekii, or Coxiella burnetii. The additivity index of two antibodies, R5E4 and R1B10 was near zero, suggesting that these two antibodies may compete for the same epitope of the 44-kD protein, while monoclonal antibody R5A9 appears to interact with a different epitope. The antibodies secreted by these hybridomas may be useful as immunologic agents in serodiagnostic, immunohistochemical, and other studies of the etiologic agent of HGE.

A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles.

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.

Altered cytochrome c display precedes apoptotic cell death in Drosophila.

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

Regulated targeting of BAX to mitochondria.

The proapoptotic protein BAX contains a single predicted transmembrane domain at its COOH terminus. In unstimulated cells, BAX is located in the cytosol and in peripheral association with intracellular membranes including mitochondria, but inserts into mitochondrial membranes after a death signal. This failure to insert into mitochondrial membrane in the absence of a death signal correlates with repression of the transmembrane signal-anchor function of BAX by the NH2-terminal domain. Targeting can be instated by deleting the domain or by replacing the BAX transmembrane segment with that of BCL-2. In stimulated cells, the contribution of the NH2 terminus of BAX correlates with further exposure of this domain after membrane insertion of the protein. The peptidyl caspase inhibitor zVAD-fmk partly blocks the stimulated mitochondrial membrane insertion of BAX in vivo, which is consistent with the ability of apoptotic cell extracts to support mitochondrial targeting of BAX in vitro, dependent on activation of caspase(s). Taken together, our results suggest that regulated targeting of BAX to mitochondria in response to a death signal is mediated by discrete domains within the BAX polypeptide. The contribution of one or more caspases may reflect an initiation and/or amplification of this regulated targeting.

B cell tolerance to a minor, but not to a major, antigenic surface of the self antigen, cytochrome c.

To study B cell tolerance to the mitochondrial protein cytochrome c (CYT), the B cell response to pigeon CYT (PCC) was examined in mice transgenic for PCC. PCC was coupled to OVA to provide T cell help, since PCC-specific T cells in PCC-transgenic mice are deleted in the thymus. The frequency of secondary B cells responding to the minor antigenic surface around residue 44 on PCC was decreased about 10-fold in native PCC-transgenic mice compared with that in control mice or in transgenic mice expressing an altered form of PCC that lacked the heme and had a different amino acid sequence at the N-terminus. A similar decrease has been observed in the frequency of B cells in normal mice recognizing the site around residue 44 on mouse CYT compared with the frequency of B cells recognizing the corresponding site on foreign CYT. There were no major decreases but apparently were compensatory increases in the frequencies of B cells recognizing other sites on PCC in the native PCC-transgenic mice compared with those in other mice. These results indicate that B cells in mice are only partially tolerant to self CYT. A possible basis for this partial tolerance relating to the fate of CYT in cell death is discussed. This may be the first example of the use of a transgenic system to study B cell tolerance to a homologous self Ag.

A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis.

Treatment of HL-60 cells with staurosporine (STS) induced mitochondrial cytochrome c efflux into the cytosol, which was followed by caspase-3 activation and apoptosis. Consistent with these observations, in vitro experiments demonstrated that, except for cytochrome c, the cytosol of HL-60 cells contained sufficient amounts of all factors required for caspase-3 activation. In contrast, treatment of HCW-2 cells (an apoptotic-resistant HL-60 subclone) with STS failed to induce significant amounts of mitochondrial cytochrome c efflux, caspase-3 activation, and apoptosis. In vitro assays strongly suggested that a lack of cytochrome c in the cytosol was the primary limiting factor for caspase-3 activation in HCW-2 cells. To explore the mechanism which regulates mitochondrial cytochrome c efflux, we developed an in vitro assay which showed that cytosolic extracts from STS-treated, but not untreated, HL-60 cells contained an activity, which we designated 'CIF' (cytochrome c-efflux inducing factor), which rapidly induced cytochrome c efflux from HL-60 mitochondria. In contrast, there was no detectable CIF activity in STS-treated HCW-2 cells although the mitochondria from HCW-2 cells were responsive to the CIF activity from STS-treated HL-60 cells. These experiments have identified a novel activity, CIF, which is required for cytochrome c efflux and they indicate that the absence of CIF is the biochemical explanation for the impaired ability of HCW-2 cells to activate caspase-3 and undergo apoptosis.

Involvement of a caspase-3-like cysteine protease in 1-methyl-4-phenylpyridinium-mediated apoptosis of cultured cerebellar granule neurons.

Exposure of various neuronal cells or cell lines to high concentrations of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), results in cell death. Recently, it has been reported that low concentrations of MPP+ induce apoptosis in susceptible neurons. We have further characterized MPP+-mediated toxicity of cultured cerebellar granule neurons (CGNs) and found that exposure of CGNs to relatively low concentrations of MPP+ results in apoptosis, whereas higher concentrations result in necrosis. Cotreatment of CGNs with MPP+ and the tetrapeptide inhibitor of caspase-3-like proteases, acetyl-DEVD-CHO, markedly attenuates apoptotic but not necrotic death of these neurons. The more specific inhibitor of caspase-1-like proteases, acetyl-YVAD-CHO, however, was ineffective against MPP+ neurotoxicity. Moreover, cytoplasmic extracts prepared from MPP+-treated CGNs contain markedly increased protease activity that cleaves the caspase-3 substrate acetyl-DEVD-p-nitroaniline. Finally, the cytoplasmic concentration of the apoptogenic protein cytochrome c was increased in a time-dependent fashion in MPP+-treated CGNs before the onset of apoptosis. Our data confirm that the neurotoxicity of MPP+ is due to both necrosis and apoptosis and suggest that the latter is mediated by activation of a caspase-3-like protease.

B lymphocyte recognition of the self antigen mouse cytochrome C in different mouse strains: targeting of the same dominant epitope by naturally-occurring cells expressing distinct VH genes.

Previously we reported that, early in the antibody response of BALB/c mice to several cytochromes c (CYT) coupled to ovalbumin (OVA), B cells responding to the self antigen mouse CYT recognized a single site on mouse CYT and were in much higher frequency than B cells responding to foreign CYT. In the present study these B cells were shown by in vitro activation of primary splenocytes to be present in naive BALB/c mice, i.e. prior to exposure to exogenous CYT. The higher frequency of B cells responsive to self versus foreign CYT was also shown in this study to occur in the early antibody response to CYT-OVA in C57BL/6 mice. The same dominant site was recognized in BALB/c mice (IgHa), C57BL/6 mice (IgHb) and the congenic strains BC-17 (IgHa on the C57BL/6 background) and CB-20 (IgHb on the BALB/c background). However, anti-mouse CYT mAbs produced in IgHb mice were shown to derive from the VH gene 5.54.4 while mAbs in IgHa mice derive from the VH gene 19.1.2. The polypeptides encoded by these VH genes, which differ by only five amino acid residues, paired with polypeptides encoded by the same Vk genes (R9 and 2G5). In both VH 19.1.2- and VH 5.54.4-derived mAbs H3 and the Vk-Jk join were variable. The affinity for mouse CYT was reduced in the VH gene 5.54.4-derived mAb due to a faster off-rate constant. This difference in affinity may relate to the lower frequency of B cells responding to mouse CYT in C57BL/6 mice than in BALB/c mice. The results show that naturally-occurring CYT-specific autoreactive B cells occur normally in more than one mouse strain and that self antigen recognition by those cells appears to be atypical involving mostly the immunoglobulin V gene-encoded segments.

Maturation of the antibody response to the major epitope on the self antigen mouse cytochrome c. Restricted V gene usage, selected mutations, and increased affinity.

Most B lymphocytes recognizing the major epitope on the self Ag mouse cytochrome c (CYT) express one particular VH gene (19.1.2) in combination with one of two Vkappa genes (R9 or 2G5). This restriction made it possible to observe changes in the primary structure of mouse CYT-specific mAb as the B cell response to mouse CYT (coupled to OVA) progressed. Thus, somatic mutations at positions 31 and 58 in the VH gene complementarity-determining regions (CDR) 1 and 2, respectively, and tyrosine at position 96 in the Vkappa-Jkappa join were frequently selected during the response. The affinity constant (ka), which increased as much as 90-fold due to a decrease in the off-rate constant, correlated with the number of somatic mutations but not strictly with the commonly selected changes. There appears to be little selection for a particular H chain CDR3 in the mouse CYT-specific mAb since it is extremely variable, in sequence and length, throughout the response. This variability may reflect that mouse CYT is a self Ag. Otherwise, the Ab response to this protein Ag is consistent with the paradigm for affinity maturation that is well established for Ab responses to haptens.

Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.

A cell-free system based on cytosols of normally growing cells is established that reproduces aspects of the apoptotic program in vitro. The apoptotic program is initiated by addition of dATP. Fractionation of cytosol yielded a 15 kDa protein that is required for in vitro apoptosis. The absorption spectrum and protein sequence revealed that this protein is cytochrome c. Elimination of cytochrome c from cytosol by immunodepletion, or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Adding back cytochrome c to the cytochrome c-depleted extracts restored their apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.

The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c.

Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags.

Major and minor epitopes on the self antigen mouse cytochrome c mapped by site-directed mutagenesis.

Variants of rat (mouse) cytochrome c, prepared by site-directed mutagenesis or represented by closely-related cytochromes c from different species, were employed to map the functional boundaries of a number of mouse monoclonal antibodies (mAb) specific for the major antigenic region on the self antigen (Ag) around residue 62 and the minor antigenic region around residue 44. The recombinant mouse cytochromes c tested were, unlike the tissue-derived Ag, trimethylated at position 72, and included the wild-type which was acetylated at the amino terminus, a variant that was unacetylated at the amino terminus, and variants with the following single amino acid residue replacements: V11I (valine to isoleucine at position 11), Q12M, A15S, A44P, F46Y, D50A, T58I and G89E. Of these, only the A44P variant affected the binding of mAb to the region previously localized to the vicinity of residue 44, thus confirming that assignment. Loss of the acetyl group at the amino terminus affected the binding of most of the mAb to the region around residue 62. The other mutations had little, if any, affect on mAb binding. The epitopes of mAb binding the region around residue 62 were shown in this study to have similar functional boundaries. This site on the self Ag, which encompasses at least three discontinuous segments of the polypeptide chain, is comparable in size to epitopes on other protein Ag that have been mapped by X-ray crystallography and is similar to an epitope in the corresponding region of the foreign Ag, horse cytochrome c, that has been mapped by hydrogen-deuterium exchange. In addition to the mAb binding the regions around residues 44 and 62, a third group of mouse cytochrome c-specific mAb known to be broadly reactive with mammalian cytochromes c and that represents a minor portion of the mAb was tested for binding the site-directed mutants of mouse cytochrome c. None of these mAb were affected by the mutations, indicating the presence of at least one more antigenic region on the self Ag in an area not encompassed by these mutations that is structurally highly conserved.

B lymphocyte recognition of cytochrome c: higher frequency of cells specific for self versus foreign antigen early in the immune response and V gene usage in the response to self antigen.

To study immunoglobulin gene usage in the antibody response of mice to the self antigen (Ag) mouse cytochrome c (cyt), B cell hybridomas were prepared from splenic B cells of immunized BALB/c mice prior to the onset of somatic mutation, i.e. 3 days after injecting ovalbumin (OVA)-primed mice with mouse cyt coupled to OVA. Monoclonal antibodies (mAb) from all of the seven primary hybridomas we obtained were sensitive to a single amino acid substitution from aspartic acid to glutamic acid at position 62 in mouse cyt. This is the specificity of the vast majority of B cells responding to mouse cyt as determined from assays of B cells activated in splenic fragment cultures. Six of the mAb derive from the 19.1.2 J558 VH gene which is also used in the response to alpha (1-->6) dextran and three of these mAb derive from the R9 V kappa gene, a member of the V kappa Ox-1 family. The other mAb derive from distinct, although similar, V kappa genes. Attempts to obtain hybridomas secreting primary (unmutated) mAb specific for cyt foreign to mice have been hampered by the much lower frequency of B cells responding early to foreign cyt in comparison to the self Ag. This suggests that, contrary to expectation of tolerance mechanisms, in naive BALB/c mice B lymphocytes specific for a single epitope on self cyt are present in higher frequency than B lymphocytes specific for similar epitopes on foreign cyt. Possible explanations for this result include biased expression in the B cell repertoire of the particular combination of V genes encoding mouse cyt-specific mAb or to positive selection of developing B lymphocytes by endogenous Ag.

Rapid refolding of native epitopes on the surface of cytochrome c.

Refolding of surface epitopes on horse cytochrome c has been measured by monoclonal antibody binding. Two antibodies were used to probe re-formation of native-like surface structure: one antibody (2B5) binds to native cytochrome c near a type II turn (residue 44) while the other (5F8) binds to a different epitope on the opposite face of the protein near the amino terminus of an alpha-helical segment (residue 60). The results show that within the first approximately 100 ms of refolding all of the unfolded protein collapses to native-like folding intermediates that contain both antibody binding sites. All three absorbance/fluorescence-detected kinetic phases in the folding of cytochrome c (k1 approximately 5 s-1, k2 approximately 0.4 s-1, k3 approximately 0.03 s-1) are slower than the rates of re-formation of the antibody binding sites (k(obs) > 10.0 s-1), suggesting that the formation of antibody binding sites precedes the refolding reactions observed in kinetically resolved optically-detected refolding phases. Kinetically unresolved folding processes account for 79% and 19% of the total fluorescence change and absorbance change, respectively, observed in equilibrium unfolding. Thus, kinetically unresolved folding reactions appear to be responsible for re-formation of the MAb binding sites within partially folded intermediate species. These species are non-native (incompletely folded) in that their optical properties are in between those of the unfolded and the fully folded protein. As a test of whether antibody binding to folding intermediate(s) perturbs further folding, the rate of the absorbance-detected slow refolding phase has been measured for folding intermediate(s) of cytochrome c complexed with antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)