Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments

Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for beta-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five 'two-state' transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures Tm and molar enthalpy changes DeltaHm gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature (Tm=36.9 and 44.1 degrees C) and two high-temperature (Tm=74.6 and 76.8 degrees C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two 'two-state' transitions for each fragment. Tm and DeltaHm values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature (Tm=67.2 and 75.9 degrees C for Fab and Fc, respectively).     

Comparative conformational studies on the tryptic digestion fragments of human immunoglobulins M and G

IgM¹ immunoglobulins were cleaved into Fabμ and (Fc)5μ fragments by tryptic digestion. Comparative circular dichroism studies with the corresponding IgG fragments show that the Fab portions of IgG and IgM proteins have very similar CD spectral features, although the same is not true for their Fc fragments. These studies indicate the presence of higher amount of beta-structured regions in Fcμ than in Fcγ. Also, there are considerable differences in their pH-dependent structural transitions as measured by CD spectral changes. The conformational differences between IgG and IgM immunoglobulins are more pronounced in their Fc portions, which carry out class specific biological functions, rather than in Fab portions, which contain antigen combining sites.     

Non-covalent interactions between Fab and Fc regions in immunoglobulin G molecules. Hydrogen-deuterium exchange studies

To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part.     

Guanidinium chloride and urea denaturations of beta-lactoglobulin A at pH 2.0 and 25 degrees C: the equilibrium intermediate contains non-native structures (helix, tryptophan and hydrophobic patches)

We have carried out guanidinium chloride (GdmCl) and urea denaturations of bovine beta-lactoglobulin A (beta-lgA) at pH 2.0 and 25 degrees C, using far-UV and near-UV circular dichroism, near-UV absorption and tryptophan fluorescence spectroscopies. The stable intermediate state that occurs during GdmCl denaturation has been characterized by the far- and near-UV circular dichroism, tryptophan difference absorption, tryptophan fluorescence and 8-anilino-1-naphthalene sulphonic acid binding measurements. Following conclusions have been reached. (a) Urea-induced denaturation is not a two-state process. (b) GdmCl-induced denaturation is composed of two distinct two-state processes. (c) alpha-Helical content, burial of tryptophan residues and burial of hydrophobic surface area are more in the GdmCl-induced stable intermediate than those originally present in the native protein.     

Circular dichroism and absorbance properties of nitrotyrosyl chromophores in staphylococcal nuclease and in a model diketopiperazine.

An active derivative of staphylococcal nuclease, in which only tyrosine residue 115 has been nitrated with use of tetranitromethane, has been characterized using absorbance, circular dichroism, and fluorescence spectroscopy. The results show that nitrotyrosine-115 nuclease is indistinguishable from native nuclease with regard to the average secondary structure of the folded polypeptide chain, the susceptibility of the enzyme to heat denaturation, and the local tertiary structure around tryptophan residue 140. Inasmuch as optical properties of nitrotyrosine-115 nuclease from 300 to 500 nm can be unambiguously assigned to nitrotyrosine residue 115 in the active site region, this modified enzyme presents a good model system for studying the circular dichroism properties of this aromatic amino acid in a protein. The spectral properties of nitrotyrosine-115 nuclease have been compared to those of the model compounds, cyclo-(-Gly-Tyr(3NO2)-) and Tyr(3NO2). Circular dichroism spectral changes in nitrotyrosine-115 nuclease due to the binding of deoxythymidine 3',5'-diphosphate and Ca-2+ have been compared to the corresponding nitrotyrosyl-115 absorption spectral changes. This comparison shows that the circular dichroism difference spectrum exhibits an over-all change in the intensity of the observed Cotton effects, whereas the absorption difference spectrum exhibits a blue shift. This finding supports the suggestion that perturbations of aromatic amino acid chromophores in proteins due to ligand binding result in red or blue shifts in absorption difference spectra, but in over-all changes of intensity in circular dichroism difference spectra.     

Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities

The single disulfide bond in potato Inhibitor I protomers (Mr = 8,000) was reduced and alkylated in the absence of denaturants to the carboxamidomethyl cysteine derivative. Two half-cystines per mol of inhibitor protomer were modified as determined by (a) loss of two sulfhydryl groups per reduced protomer, (b) incorporation of 2 mol of [14C]iodoacetamide per protomer, and (c) amino acid analyses. The alkylated Inhibitor I retained its oligomeric structure (Mr = 40,000) and fully retained its immunological cross reactivity with anti-Inhibitor I serum. Furthermore, the inhibitory properties of modified and unmodified Inhibitor I toward chymotrypsin were identical. The stoichiometric complex between modified Inhibitor I and chymotrypsin was stable at pH 8.0 for over 72 h. The modification of the disulfide bond introduced a lability to both heat and proteolytic enzymes. Ultraviolet difference spectra and near ultraviolet circular dichroism spectra at pH 8.0, between modified and unmodified Inhibitor I, revealed only minor absorption changes due to modification. However, in circular dichroism spectra at pH 2.0, the modified inhibitor exhibited a significant loss of absorption in the region between 270 and 280 nm that was present in the unmodified inhibitor. The cumulative results of this study indicate that the single disulfide bond in Inhibitor I, which forms a rather large disulfide loop between residues 5 and 51, is not important at neutral pH for maintaining major structural conformations which affect immunological or inhibitory activities, but the disulfide bond does impose restraints on the protein which stabilize it toward thermal denaturation and proteolysis. The susceptibility of the reduced inhibitor to plant sulfhydryl enzymes may have some importance in in vivo degradation.     

Effect of salt concentration on immunoglobulin G structure

An effect of salt concentration on the human myeloma immunoglobulin G structure was studied by means of circular dichroism, thermal perturbation difference spectroscopy and isoelectric focusing in a pH gradient created by a concentration gradient of glucose in borate buffer solution. Immunoglobulin G (K) Iva showed a significant shift of isoelectric point to the alkaline region as a result of the increase in salt concentration. The difference spectra indicated a change in the exposure of tyrosine residues as a result of increase in salt concentration. No changes in the circular dichroic spectra with salt concentration were observed between 205 and 250 nm. Spectral changes observed for the undigested immunoglobulin G molecule are more marked than those observed for the isolated Fab fragments.     

Circular dichroism and conformation of human alpha1-antitrypsin.

The solution conformation of alpha 1-antitrypsin from human blood plasma was studied by the circular dichroism (CD) probe. The CD spectra revealed in this glycoprotein approximately 16-20% of alpha-helix, the rest of the main polypeptide chain possessing the pleated sheet (beta) and the aperiodic structures. The conformation was stable between pH 4.7 and 8.8. Reversible change in conformation was observed at pH 10.3, and more dratic denaturation occurred at pH 11.6. The environment of the side chain chromophores was strongly affected by acid at pH 2.5, whereas the main chain conformation was changed slightly. A drastic change in the CD spectra, indicating denaturation, was observed in 3.5 M guanidine hydrochloride. Sodium dodecyl sulfate was effective in disorganizing the tertiary structure and in enhancing the helix content. The phenylalanine band fine structure was observed in the native protein and also after denaturation with acid, guanidine hydrochloride and sodium dodecyl sulfate.     

Recognition of polynucleotides by antibodies to poly(I), poly(C).

The binding of anti poly(I). poly (C) Fab fragments to double or triple stranded polynucletides has been studied by fluorescence. Association constants were deduced from competition experiments. The comparison of the association constants leads to the conclusion that several atoms of the base residues do not interact with the amino acid residues of the binding site of Fab fragment while the hydroxyl groups of furanose rings interact. These results suggest that the Fab fragments do not bind to the major groove of the double stranded polynucleotides. An interaction between the C(2)O group of pyrimidine residues and Fab fragments cannot be excluded. Circular dichroism of poly(I). poly(C) or poly(I). poly(br5C)-Fab fragments complexes are very different from the circular dichroism of free polynucleotides which suggests a deformation of the polynucleotides bound to the Fab fragments.     

Temperature and solvent effects on reaction centers from Chloroflexus aurantiacus and Chromatium tepidum.

Temperature and solvent effects on reaction center structures were examined in two thermophilic photosynthetic bacteria, Chloroflexus aurantiacus and Chromatium tepidum, in order to gain insight into the interactions among the reaction center proteins and pigment systems. Thermal stability of the reaction centers was found to be proportional to the optimum growth temperature. Circular dichroism (CD) spectra in the 250-300 nm region indicated that thermal denaturation destroyed tertiary structures (helix-to-helix interactions or amino acid residue conformation) in the native reaction center, keeping helical structures intact. Absorption and circular dichroism spectral changes showed that alcohol denatured the so-called special pair and the accessory BChl a independently. The alcohol denaturation further indicates that the coordination between BChl a and amino acid residue in the protein is one of the important interactions maintaining the pigment organization of the reaction centers.     

Binding of m-nitrobenzeneboronic acid to the active site of subtilisin BPN.

m-Nitrobenzeneboronic acid as a possible transition-state analog for serine proteases was found to cause absorption spectral change from 250 nm 350 nm upon binding with subtilisin BPN' (EC 3.4.21.14) at pH 6.5. Similar difference spectral changes of m-nitrobenzeneboronic acid were also observed at alkaline pH or upon addition of N-methylimidazole at pH 6.5. A characteristic circular dichroism spectrum of m-nitrobenezeneboronic acid was induced upon binding with subtilisin BPN' not only at pH 6.5, but also at alkaline pH. Circular dichroism spectral titration confirmed the stoichiometry of 1 : 1 for the m-nitrobenzeneboronic acid - subtilisin complex. m-Nitrobenzeneboronic acid was shown to be useful as a reversible chromophoric probe for the catalytic site of serine proteases.     

Native-like tertiary structure in the Mucor miehei lipase molten globule state obtained at low pH

Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy and binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid-unfolded state approximately pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits characteristics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native-like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native-like tertiary structure is an interesting observation made for this lipase.     

More stable structure of wheat germ lipase at low pH than its native state

Wheat germ lipase is a cereal lipase which is a monomeric protein. In the present study we sought to structurally characterize this protein along with equilibrium unfolding in solution. Conformational changes occurring in the protein with varying pH, were monitored by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy, binding of hydrophobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS) and dynamic light scattering (DLS). Our study showed that acid denaturation of lipase lead to characterization of multiple monomeric intermediates. Native protein at pH 7.0 showed far-UV spectrum indicating mixed structure with both alpha and beta-type of characteristics. Activity of lipase was found to fall on either sides of pH 7.0–8.0. Acid-unfolded state was characterized at pH 4.0 with residual secondary structure, disrupted tertiary spectrum and red-shifted fluorescence spectrum with decreased intensity. Further decrease in pH lead to formation of secondary structure and acid-induced molten globule state was found to be stabilized at pH 1.4, with exposed tryptophan residues and hydrophobic patches. Notably, interesting finding of this study was characterization of acid-induced state at pH 0.8 with higher secondary structure content than native lipase, regain in tertiary spectrum and induction of compact conformation. Although enzymatically inactive, acid-induced state at pH 0.8 was found to be structurally more stable than native lipase, as shown by chemical and thermal denaturation profiles.     

Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen.

Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.     

Effects of acid pH and urea on the spectral properties of the LHII antenna complex from the photosynthetic bacterium Ectothiorhodospira sp.

The aim of this study was to investigate the spectral modifications of the LHII antenna complex from the purple bacterium Ectothiorhodospira sp. upon acid pH titration both in the presence and absence of urea. A blue shift specifically and reversibly affected the B850 band around pH 5.5-6.0 suggesting that a histidine residue most probably participated in the in vivo absorption red shifting mechanism. This transition was observed in the presence and absence of urea. Under strong chaotropic conditions, a second transition occurred around pH 2.0, affecting the B800 band irreversibly and the B850 reversibly. Under these conditions a blue shift from 856 to 842 nm occurred and a new and strong circular dichroism signal from the new 842 nm band was observed. Reverting to the original experimental conditions induced a red shift of the B850 band up to 856 nm but the circular dichroism signal remained mostly unaffected. Under the same experimental conditions, i.e. pH 2.1 in the presence of urea, part of the B800 band was irreversibly destroyed with concomitant appearance of a band around 770 nm due to monomeric bacteriochlorophyll from the disrupted B800. Furthermore, Gaussian deconvolution and second derivative of the reverted spectra at pH 8.0 after strong-acid treatment indicated that the new B850 band was actually composed of two bands centered at 843 and 858 nm. We ascribed the 858 nm band to bacteriochlorophylls that underwent reversible spectral shift and the 843 nm band to oligomeric bacteriopheophytin formed from a part of the B850 bacteriochlorophyll. This new oligomer would be responsible for the observed strong and mostly conservative circular dichroism signal. The presence of bacteriopheophytin in the reverted samples was definitively demonstrated by HPLC pigment analysis. The pheophytinization process progressed as the pH decreased below 2.1, and at a certain point (i.e. pH 1.5) all bacteriochlorophylls, including those from the B800 band, became converted to oligomeric bacteriopheophytin, as shown by the presence of a single absorption band around 843 nm and by the appearance of a single main elution peak in the HPLC chromatogram which corresponded to bacteriopheophytin.     

Examining the relationship between secondary structure and antibody recognition in immunopeptides from foot-and-mouth disease virus

Summary The conformation of a peptide that represents antigenic site A of foot-and-mouth disease virus strain C-S8c1 (residues 136–156 of VP1; YTASARGDLAHLTTTHARHLP) has been studied by circular dichroism and compared with three analogs that reproduce amino acid substitutions at position 146 (HisArg, Gln or Asp) which affect antibody recognition. Four other peptides, incorporating replacements at position 147 predicted to maintain (LeuIle, Nle and Ala) or disrupt (LeuGly) helical structure at this site, have also been studied. In aqueous solution or in 4 M urea, the spectra of all eight peptides were typical of aperiodic conformation and independent of concentration or pH. However, upon addition of solvents such as methanol or hexafluoroisopropanol, spectral patterns evidenced significant levels (ca. 50%) of helical structure. The single residue substitutions at positions 146 and 147 caused minor to significant variations in the calculated amount of -helix of the peptides. An attempt to relate these changes in helical content to the antigenic behaviour of the peptides towards five monoclonal antibodies elicited with virus and mapping at site A could not find any straightforward correspondence between the two sets of results. The parent peptide and its His¹⁴⁶Arg analog were also analyzed by circular dichroism in the presence of the Fab fragment of SD6, a monoclonal antibody mapping at site A and much less reactive with viruses carrying the referred mutation. Although a peptide-antibody interaction was evident from spectral changes, careful inspection of the difference spectra (peptide-Fab minus Fab) of both peptides failed to detect any significant distinction between them that could be attributed to their different immunoreactivity. While these findings do not necessarily conflict with previous reports that the interaction of antigenic site A with antibodies is mediated to some extent by the adoption of a helix structure, they suggest that, at least for C-serotype viruses, other structural features in addition to a helical conformation are critically involved in antigenic recognition.     

Stability of the allergenic soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a 21.5 kDa allergenic protein that belongs to the family of all antiparallel beta-sheet proteins that are highly resistant to thermal and chemical denaturation. Spectroscopic and biochemical techniques such as circular dichroism (CD), ANS fluorescence and proteolysis were used to study its molecular structure under denaturing conditions such as acid and heat to which these allergens are commonly exposed during food processing. Reduction of native SKTI leads to its complete and rapid proteolysis by pepsin in simulated gastric fluid (SGF). Limited proteolysis with chymotrypsin during renaturation after heating showed that the native structure reforms at around 60 degrees C reversing the denaturation. CD spectra revealed that under acid denaturing conditions, SKTI shows major changes in conformation, indicating the possibility of a molten structure. The existence of this intermediate was established by ANS fluorescence studies at different concentrations of HCl. The remarkable stability of SKTI to both thermal and acid denaturation may be important for its role as a food allergen.     

The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein

The denaturation of immunoglobulin G was studied by different calorimetric methods and circular dichroism spectroscopy. The thermogram of the immunoglobulin showed two main transitions that are a superimposition of distinct denaturation steps. It was shown that the two transitions have different sensitivities to changes in temperature and pH. The two peaks represent the F(ab) and F(c) fragments of the IgG molecule. The F(ab) fragment is most sensitive to heat treatment, whereas the F(c) fragment is most sensitive to decreasing pH. The transitions were independent, and the unfolding was immediately followed by an irreversible aggregation step. Below the unfolding temperature, the unfolding is the rate-determining step in the overall denaturation process. At higher temperatures where a relatively high concentration of (partially) unfolded IgG molecules is present, the rate of aggregation is so fast that IgG molecules become locked in aggregates before they are completely denatured. Furthermore, the structure of the aggregates formed depends on the denaturation method. The circular dichroism spectrum of the IgG is also strongly affected by both heat treatment and low pH treatment. It was shown that a strong correlation exists between the denaturation transitions as observed by calorimetry and the changes in secondary structure derived from circular dichroism. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.     

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.     

Alkalin titrations of human somatotropin, human choriomammotropin and ovine prolactin by circular dichroism and fluorescence.

Structural transitions occurring during the alkalin titration of human somatotropin, human choriomammotropin, and ovine prolactin have been investigated by means of circular dichroism and fluorescence emission spectra. Human somatotropin exhibited an isodichroic point at 287 nm, with all spectral changes being reversed upon back titration from pH 12.50 to pH 8.0. Fluorescence quenching as a function of pH produced a simple sigmoidal curve. Human choriomammotropin exhibited an isodichroic point at 288 nm. The fluorescence and circular dichroism spectra of this protein were found to be reversible between pH 8.0 and 11.0. However, on titration above pH 11, the isodichroic point and the reversibility of the circular dichroism spectra were lost. This conformational transition was accompanied by a sharp increase in fluorescence quantum yield. The circular dichroism spectra of ovine prolactin showed essentially no change on titration to pH 11.0. However, between pH 11.0 and 12.0, a sharp conformational transition was observed similar to that seen in human choriomammotropin, but not exhibiting the same increase in fluorescence quantum yield. The fluorescence titration of prolactin was found to be essentially reversible upon back titration from pH 12.5, although the circular dichroism spectra were not reversible from this pH.