Effects of the stabilization of the molten globule state on the folding mechanism of alpha-lactalbumin: a study of a chimera of bovine and human alpha-lactalbumin

The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.     

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat,trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis

The calcium-depleted form of alpha-lactalbumin (alpha-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-alpha-LA at neutral pH. CD spectra revealed that the molten globule of apo-alpha-LA can be obtained upon mild heating at 45 degrees C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far- and near-UV CD spectra of apo-alpha-LA are essentially identical to those of the most studied molten globule of alpha-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-alpha-LA by proteinase K at 4 degrees C occurs slowly as an all-or-none process leading to small peptides only. At 37 degrees C, proteinase K preferentially cleaves apo-alpha-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the beta-subdomain of the protein (chain region 34-57). Similar sites of preferential cleavage have been observed with the TFE- and oleic acid-induced molten globule of apo-alpha-LA. A protein species given by the N-terminal fragment 1-34 linked via the four disulfide bridges to the C-terminal fragment 54-123 or 57-123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-alpha-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of alpha-LA maintains a native-like tertiary fold characterized by a rather well-structured alpha-domain and a disordered chain region encompassing the beta-subdomain 34-57 of the protein.     

A comparative study of the alpha-subdomains of bovine and human alpha-lactalbumin reveals key differences that correlate with molten globule stability

The alpha-lactalbumins form stable molten globule states under a range of conditions, with the low pH form being the best characterized. The stability of the molten globule varies among different members of this family, but the origin of the stability difference is not clear. We compare the folding and stability of alpha-subdomain constructs of human and bovine alpha-lactalbumin. Previous studies have demonstrated that the isolated alpha-subdomain of human alpha-lactalbumin folds and forms a molten globule state. The minimum core construct has been defined to include the A, B, and D alpha-helices and the C-terminal 3(10) helix. A construct corresponding to the same region of bovine alpha-lactalbumin is much less structured and less stable than the human alpha-lactalbumin construct. Addition of the C-helix to generate a 75-residue bovine construct does not lead to a significant increase in structure or stability. This construct (AB-CD/3(10)) contains the entire alpha-subdomain of bovine alpha-lactalbumin. Thus molten globule formation in the human protein, but not in the bovine protein, can be rationalized on the basis of a stable alpha-subdomain. Interactions involving more of the protein chain are required to generate a well structured molten globule in the bovine protein. Comparison of AB-CD/3(10) to the molten globule formed by the intact protein and to the protein with the 6-120 disulfide reduced indicates that both the beta-subdomain and the 6-120 disulfide play a role in stabilizing the bovine alpha-lactalbumin molten globule.     

1,1,1,3,3,3-hexafluoroisopropanol induced thermal unfolding and molten globule state of bovine alpha-lactalbumin: calorimetric and spectroscopic studies

The thermal denaturation of alpha-lactalbumin was studied at pH 7.0 and 9.0 in aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) by high-sensitivity differential scanning calorimetry. The conformation of the protein was analyzed by a combination of fluorescence and circular dichroism measurements. The most obvious effect of HFIP was lowering of the transition temperature with an increase in the concentration of the alcohol up to 0.30M, beyond which no calorimetric transition was observed. Up to 0.30M HFIP the calorimetric and van't Hoff enthalpy remained the same, indicating the validity of the two-state approximation for the thermal unfolding of alpha-lactalbumin. The quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. Spectroscopic observations confirm that alpha-lactalbumin is in the molten globule state in the presence of 0.50M HFIP at pH 7.0 and 0.75M HFIP at pH 9.0. The results also demonstrate that alpha-lactalbumin in the molten globule state undergoes a noncooperative thermal transition to the denatured state. It is observed that two of four tryptophans are exposed to the solvent in the HFIP induced molten globule state of alpha-lactalbumin compared to four in the 8.5M urea induced denatured state of the protein. It is also observed that the HFIP induced molten globule states at the two pH values are different from the acid induced molten globule state (A state) of alpha-lactalbumin.     

Structural characterisation of the human alpha-lactalbumin molten globule at high temperature

Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments.     

Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering.

A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the alpha-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of alpha-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of alpha-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the alpha-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the alpha-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.     

Effect of hydrostatic pressure on unfolding of alpha-lactalbumin: volumetric equivalence of the molten globule and unfolded state

The effect of pressure on the unfolding of bovine alpha-lactalbumin was investigated by ultraviolet absorption methods. The change of molar volume associated with unfolding, deltaV, was measured in the presence or absence of guanidine hydrochloride at pH 7. The deltaV was estimated to be -63 cm3/mol in the absence of a chemical denaturant. While in the presence of guanidine hydrochloride (GuHCl), it was found that deltaV was -66 cm3/mol at 25 degrees C and was independent of the concentration of GuHCl, despite the fact that the molten globule fraction in the total unfolding product decreased with the increase of GuHCl concentration. The results indicate that the volume of alpha-lactalbumin only changes at the transition from a native to a molten globule state, and almost no volume change has been found during the transition from a molten globule to the unfolded state.     

Local and global cooperativity in the human alpha-lactalbumin molten globule

NMR spectroscopy has been used to follow the urea-induced unfolding of the low pH molten globule states of a single-disulfide variant of human alpha-lactalbumin ([28-111] alpha-LA) and of two mutants, each with a single proline substitution in a helix. [28-111] alpha-LA forms a molten globule very similar to that formed by the wild-type four-disulfide protein, and this variant has been used as a model for the alpha-lactalbumin (alpha-LA) molten globule in a number of studies. The urea-induced unfolding behavior of [28-111] alpha-LA is similar to that of the four-disulfide form of the protein, except that [28-111] alpha-LA is less stable and has greater cooperativity in the loss of different elements of structure. For one mutant, L11P, the helix containing the mutation is highly destabilized such that it is completely unfolded even in the absence of urea. By contrast, for the other mutant, Q117P, the helix containing the mutation retains its compact structure. Both mutations, however, show significant long-range destabilization of the overall fold showing that the molten globule state has a degree of global cooperativity. The results reveal that different permutations of three of the four major alpha-helices of the protein can form a stable, locally cooperative, compact structural core. Taken together, these findings demonstrate that the molten globule state of alpha-LA is an ensemble of conformations, with different subsets of structures linked by a range of long-range interactions.     

A model of dynamic side-chain--side-chain interactions in the alpha-lactalbumin molten globule

Proteins in the molten globule state contain high levels of secondary structure, as well as a rudimentary, nativelike tertiary topology. Thus, the structural similarity between the molten globule and native proteins may have a significant bearing in understanding the protein-folding problem. To explore the nature of side-chain--side-chain interactions in the alpha-lactalbumin (alpha-LA) molten globule, we determined the effective concentration for formation of the 28--111 disulfide bond in 14 double-mutant proteins, each containing two hydrophobic core residues replaced by alanine. We compared our results with those of single-alanine substitutions using the framework of double-mutant cycle analysis and found that, in the majority of cases, the effects of two alanine substitutions are additive. Based on these results, we propose a model of side-chain-side-chain interactions in the alpha-LA molten globule, which takes into consideration the dynamic nature of this partially folded species.     

Characterization of the denaturation of human alpha-lactalbumin in urea by molecular dynamics simulations

Molecular dynamics (MD) simulations were used to characterize the non-cooperative denaturation of the molten globule A-state of human alpha-lactalbumin by urea. A solvent of explicit urea and water molecules was used, corresponding to a urea concentration of approximately 6M. Three simulations were performed at temperatures of 293K, 360K and 400K, with lengths of 2 ns, 8 ns and 8 ns respectively. The results of the simulations were compared with experimental data from NMR studies of human alpha-lactalbumin and related peptides. During the simulations, hydrogen bonds were formed from the protein to both urea and water molecules as intra-protein hydrogen bonds were lost. Urea was shown to compete efficiently with water as both a hydrogen bond donor and acceptor. Radial distribution functions of water and urea around hydrophobic side chain atoms showed a significant increase in urea molecules in the solvation shell as the side chains became exposed during denaturation. A considerable portion of the native-like secondary structure persisted throughout the simulations. However, in the simulations at 360K and 400K, there were substantial changes in the packing of aromatic and other hydrophobic side chains in the protein, and many native contacts were lost. The results suggest that during the non-cooperative denaturation of the molten globule, secondary structure elements are stabilized by non-specific, non-native interactions.     

Effects of a helix substitution on the folding mechanism of bovine alpha-lactalbumin

The structure, stability, and unfolding-refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105-110) comes from equine lysozyme and the remainder of the sequence comes from bovine alpha-lactalbumin were studied by circular dichroism spectroscopy and stopped-flow measurements, and the results were compared with those of bovine alpha-lactalbumin. The substitution of the flexible loop in bovine alpha-lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine alpha-lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine alpha-lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non-native interaction in the flexible loop region and that the disruption of the non-native interaction reduces the free energy barrier of refolding. We conclude that the non-native interaction in the molten globule state may act as a kinetic trap for the folding of alpha-lactalbumin.     

Structural energetics of the molten globule state

Certain partly ordered protein conformations, commonly called “moltenglobule states,” are widely believed to represent protein folding intermediates. Recentstructural studies of molten globule states ofdifferent proteins have revealed features whichappear to be general in scope. The emergingconsensus is that these partly ordered forms exhibit a high content of secondary structure, considerable compactness, nonspecific tertiary structure, and significant structural flexibility. These characteristics may be used to define ageneral state of protein folding called “the molten globule state,” which is structurally andthermodynamically distinct from both the native state and the denatured state. Despite exaatensive knowledge of structural features of afew molten globule states, a cogent thermodynamic argument for their stability has not yetbeen advanced. The prevailing opinion of thelast decade was that there is little or no enthalpy difference or heat capacity differencebetween the molten globule state and the unfolded state. This view, however, appears to beat variance with the existing database of protein structural energetics and with recent estimates of the energetics of denaturation of α-lactalbumin, cytochrome c, apomyoglobin, and T4 lysozyme. We discuss these four proteins at length. The results of structural studies, together with the existing thermodynamic values for fundamental interactions in proteins, provide the foundation for a structural thermodynamic framework which can account for the observed behavior of molten globule states. Within this framework, we analyze the physical basis for both the high stability of several molten globule states and the low probability of other protential folding intermediates. Additionally, we consider, in terms of reduced enthalpy changes and disrupted cooperative interactions, the thermodynamic basis for the apparent absence of a thermally induced, cooperative unfolding transition for some molten globule states. © 1993 Wiley-Liss, Inc.     

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.     

Transition state in the folding of alpha-lactalbumin probed by the 6-120 disulfide bond.

The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of alpha-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact alpha-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of alpha-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of alpha-lactalbumin is discussed.     

Folding of bovine growth hormone is consistent with the molten globule hypothesis

Previous results from equilibrium and kinetic studies of the folding of bovine growth hormone (bGH) have demonstrated that bGH does not follow a simple two-step folding mechanism. These results are summarized and interpreted according to the "molten globule" model. The molten globule state of bGH is characterized as a folding intermediate which is largely alpha-helical, retains a compact hydrodynamic radius, has packing of the aromatic side chains that is similar to the unfolded state, and possesses a solvent-exposed hydrophobic surface along helix 106-127 that readily leads to association.     

Hydrophobic photolabeling as a new method for structural characterization of molten globule and related protein folding intermediates.

Recent advances in attempts to unravel the protein folding mechanism have indicated the need to identify the folding intermediates. Despite their transient nature, in a number of cases it has been possible to detect and characterize some of the equilibrium intermediates, for example, the molten globule (MG) state. The key features of the MG state are retention of substantial secondary structure of the native state, considerable loss of tertiary structure leading to increased hydrophobic exposure, and a compact structure. NMR, circular dichroism, and fluorescence spectroscopies have been most useful in characterizing such intermediates. We report here a new method for structural characterization of the MG state that involves probing the exposed hydrophobic sites with a hydrophobic photoactivable reagent--2[3H]diazofluorene. This carbene-based reagent binds to hydrophobic sites, and on photolysis covalently attaches itself to the neighboring amino acid side chains. The reagent photolabels alpha-lactalbumin as a function of pH (3-7.4), the labeling at neutral pH being negligible and maximal at pH 3. Chemical and proteolytic fragmentation of the photolabeled protein followed by peptide sequencing permitted identification of the labeled residues. The results obtained indicate that the sequence corresponding to B (23-34) and C (86-98) helix of the native structure are extensively labeled. The small beta-domain (40-50) is poorly labeled, Val42 being the only residue that is significantly labeled. Our data, like NMR data, indicate that in the MG state of alpha-lactalbumin, the alpha-domain has a greater degree of persistent structure than the beta-domain. However, unlike the NMR method, the photolabeling method is not limited by the size of the protein and can provide information on several new residues, for example, Leu115. The current method using DAF thus allows identification of stable and hydrophobic exposed regions in folding intermediates as the reagent binds and on photolysis covalently links to these regions.     

Equilibrium phi-analysis of a molten globule: the 1-149 apoflavodoxin fragment

The apoflavodoxin fragment comprising residues 1-149 that can be obtained by chemical cleavage of the C-terminal alpha-helix of the full-length protein is known to populate a molten globule conformation that displays a cooperative behaviour and experiences two-state urea and thermal denaturation. Here, we have used a recombinant form of this fragment to investigate molten globule energetics and to derive structural information by equilibrium Phi-analysis. We have characterized 15 mutant fragments designed to probe the persistence of native interactions in the molten globule and compared their conformational stability to that of the equivalent full-length apoflavodoxin mutants. According to our data, most of the mutations analysed modify the stability of the molten globule fragment following the trend observed when the same mutations are implemented in the full-length protein. However, the changes in stability observed in the molten globule are much smaller and the Phi-values calculated are (with a single exception) below 0.4. This is consistent with an overall and significant debilitation of the native structure. Nevertheless, the fact that the molten globule fragment can be stabilised using as a guide the native structure of the full-length protein (by increasing helix propensity, optimising charge interactions and filling small cavities) suggests that the overall structure of the molten globule is still quite close to native, in spite of the lowered stability observed.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

Kinetics of interaction of partially folded proteins with a hydrophobic dye: evidence that molten globule character is maximal in early folding intermediates.

Interaction with 8-anilino-1-naphthalenesulfonate (ANS) is widely used to detect molten globule states of proteins. We have found that even with stable partially folded states, the development of the fluorescence enhancements resulting from such interactions can be relatively slow and kinetically complex. This is probably because initial binding of the dye can induce subsequent changes in the protein structure, so that the ultimate resulting fluorescence enhancement is not necessarily a good, nonperturbing probe of the preexisting state of the protein. When ANS is used to study folding mechanisms the problem is compounded by the difficulty of distinguishing effects due to the development of dye interactions from those due to the changing populations of folding intermediates. Many of these complications can be avoided by experiments where the ANS is introduced only after folding has been allowed to proceed for a variable time. The initial fluorescence intensity after mixing, resulting only from rapid and therefore hopefully relatively nonperturbing interactions with the protein, can be monitored at different refolding times to provide a better reflection of the progress of the reaction, uncomplicated by dye interaction effects. Such studies of the folding of carbonic anhydrase and alpha-lactalbumin have been compared with conventional single-mix experiments and large discrepancies observed. When ANS was present throughout refolding, time-dependent changes attributed to the formation or reorganization of protein-ANS complexes were clearly superimposed on those associated with the actual progress of refolding, and the folding kinetics and population of intermediates were also substantially perturbed by the dye. Thus, it is clear that the pulse method, though cumbersome, should be used where refolding reactions are to be probed by dye binding. The results emphasize that fluorescence enhancement tends to be greatest in early intermediates, in contrast to what, for carbonic anhydrase at least, might appear to be the case from the more conventional experiments. Later intermediates in the folding of both of these proteins actually induce little fluorescence enhancement and therefore may be quite different in nature from equilibrium molten globule states.