Body mass index and depressive symptoms in adolescents in Taiwan: testing mediation effects of peer victimization and sleep problems.

BACKGROUND: Despite recognition of the link between body mass index (BMI) and depression in adolescence, the underlying mechanisms behind this association remain understudied. This study aims to examine three mediational pathways from BMI to depressive symptoms through peer victimization and sleep problems. Sex differences in the mediating effects were also explored. METHODS: Data came from 1893 adolescents participating in a multi-wave longitudinal study from grade 9 to 12 in northern Taiwan were analyzed. Measures included BMI in 2009, peer victimization in 2010, sleep problems in 2011, depressive symptoms in 2012 and other covariates (sex, age, parental education, family structure, family economic stress, stressful life events, pubertal development and previous scores of focal study variables). A series of multiple regression models were conducted to test mediation hypotheses. A bootstrapping approach was applied to obtain confidence intervals for determining the significance of indirect effects. RESULTS: The association between BMI and depressive symptoms was significantly mediated by peer victimization and sleep problems. Higher BMI predicted more peer victimization and sleep problems, each of which led to higher levels of depressive symptoms. Our results further showed that higher BMI was associated with more peer victimization, which led to greater sleep problems and in turn resulted in increased depressive symptoms. No sex differences was found for the indirect effects of BMI on depressive symptoms through either peer victimization or sleep problems. CONCLUSIONS: Peer victimization and sleep problems partly explain the link between BMI and depressive symptoms. Interventions to prevent or manage depressive symptoms may yield better results if they consider the effects of these two psychosocial factors rather than targeting BMI alone.

A new ultrasensitive scanning calorimeter.

A new ultrasensitive differential scanning calorimeter is described, having a number of novel features arising from integration between hardware and software. It is capable of high performance in either a scanning or isothermal mode of operation. Upscanning is carried out adiabatically while downscanning is nonadiabatic. By using software-controlled signals sent continuously to appropriate hardware devices, it is possible to improve adiabaticity and constancy of scan rate through use of empirical prerun information stored in memory rather than by using feedback systems which respond in real time and generate thermal noise. Also, instrument response time is software-selectable, maximizing performance for both slow- and fast-transient systems. While these and other sophisticated functionalities have been introduced into the instrument to improve performance and data analysis, they are virtually invisible and add no additional complexities into operation of the instrument. Noise and baseline repeatability are an order of magnitude better than published raw data from other instruments so that high-quality results can be obtained on protein solutions, for example, using as little as 50 microg of protein in the sample cell.

Receptor recognition sites reside in both lobes of human serum transferrin.

The binding of iron by transferrin leads to a significant conformational change in each lobe of the protein. Numerous studies have shown that the transferrin receptor discriminates between iron-saturated and iron-free transferrin and that it modulates the release of iron. Given these observations, it seems likely that there is contact between each lobe of transferrin and the receptor. This is the case with chicken transferrin, in which it has been demonstrated unambiguously that both lobes are required for binding and iron donation to occur [Brown-Mason and Woodworth (1984) J. Biol. Chem. 259, 1866-1873]. Further support to this contention is added by the ability of both N- and C-domain-specific monoclonal antibodies to block the binding of a solution containing both lobes [Mason, Brown and Church (1987) J. Biol. Chem. 262, 9011-9015]. In the present study a similar conclusion is reached for the binding of human serum transferrin to the transferrin receptor. With the use of recombinant N- and C-lobes of human transferrin produced in a mammalian expression system, we show that both lobes are required to achieve full binding. (Production of recombinant C-lobe in the baby hamster kidney cell system is reported here for the first time.) Each lobe is able to donate iron to transferrin receptors on HeLa S3 cells in the presence of the contralateral lobe. The results are not identical with the chicken system, because the C-lobe alone shows a limited ability to bind to receptors and to donate iron. Further complications arise from the relatively weak re-association between the two lobes of human transferrin compared with the re-association of the ovotransferrin lobes. However, domain-specific monoclonal antibodies to either lobe block the binding of N- and C-lobe mixtures in the human system, thus substantiating the need for both.

[Clinical study on ligustrazine in treating myocardial ischemia and reperfusion injury].

OBJECTIVE: To explore the protective effect of Ligustrazine in treating myocardial ischemia, and reperfusion injury. METHODS: The activities of serum superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and lactic dehydrogenase (LDH) and the amount of malondialdehyde (MDA) as well as the effects of Ligustrazine (LGT) on them were determined in 16 patients with cardiopulmonary bypass, who were, scheduled for elective cardiac surgery, were randomly divided into control group and LGT group. Ligustrazine was given by intravenous drip within 2-3 minute with a definite speed before occlusion and immediately after release respectively. Their venous blood samples were collected to measure the serum levels of SOD, GSH-Px, LDH and MDA by biochemical methods before the occlusion of aorta, at 30 minutes of occlusion and at 30 minutes after release respectively. RESULTS: There were significantly and very significantly differences between the values of control group and LGT group. CONCLUSION: LGT could effectively protect the myocardium from ischemia and reperfusion injury.

Association of the two lobes of ovotransferrin is a prerequisite for receptor recognition. Studies with recombinant ovotransferrins.

Different recombinant N-lobes of chicken ovotransferrin (oTF/2N) have been isolated from the tissue-culture medium of baby hamster kidney cells transfected with the plasmid pNUT containing the relevant DNA coding sequence. Levels of up to 40, 55 and 30 mg/1 oTF/2N were obtained for constructs defining residues 1-319, 1-332 and 1-337-(Ala)3 respectively. In addition, a full-length non-glycosylated oTF was expressed at a maximum of 80 mg/1 and a foreshortened oTF consisting of residues 1-682 was expressed at a level of 95 mg/l. These preparations were then used to produce, proteolytically, two different C-lobes (oTF/2C) comprising residues 342-686 and 342-682. The purified recombinant N-lobes (oTF/2N) are similar to the proteolytically derived half-molecule with regard to immunoreactivity and spectral properties; they show some interesting differences in thermal stability. A sequence analysis of the cDNA revealed six changes at the nucleotide level that led to six differences in the amino acid sequence compared with that reported by Jeltsch and Chambon [(1982) Eur. J. Biochem. 122, 291-295]. Electrospray mass spectrometry gives results consistent with these six changes. Interaction between the various N- and C-lobes was measured by titration calorimetry. Studies show that only those lobes that associate in solution are able to bind to the receptors on chick embryo red blood cells. These findings do not support a previous report by Oratore et al.

Production and isolation of the recombinant N-lobe of human serum transferrin from the methylotrophic yeast Pichia pastoris.

The N-lobe of human serum transferrin has been expressed in the methylotrophic yeast Pichia pastoris by placing the hTF/2N cDNA under the control of the methanol-inducible alcohol oxidase promoter. Following induction with methanol, the N-lobe was efficiently secreted into a basal salt medium in shake flasks at a level of 150-240 mg/liter. As judged by mobility on SDS-PAGE, immunoreactivity with two domain-specific monoclonal antibodies, and both thermal stability and spectral properties (indictative of correct folding and ability to bind iron), the recombinant N-lobe produced by the yeast cells appears to be identical to that produced in a mammalian expression system. Electrospray-mass spectrometry and a third domain specific antibody, however, show that approximately 80% of the protein from the yeast cells contains one or two hexose residues.

Effects of C-terminal deletions on the conformational state and denaturation of phosphoglycerate kinase.

Phosphoglycerate kinase (PGK) contains two domains of approximately equal size, both of the alpha/beta type. An alpha-helix consisting of the middle section of the 415-amino acid polypeptide chain, and the N- and C-termini reside in the interdomain hinge region [Watson, H. C., et al. (1982) EMBO J. 1, 1635-1640]. The C-terminal end is an integral part of the N-terminal domain. The consequences of the deletion of fifteen and three C-terminal amino acids on the conformational state and on the guanidine hydrochloride-induced and thermal unfolding of PGK were investigated by using near- and far-UV CD, tryptophan fluorescence, 1-anilinonaphthalene-8-sulfonic acid binding, accessibility to chemical modification, and differential scanning calorimetry. The results of these studies indicate that the conformations of both domains and of the interdomain region were altered by these deletions. In the absence of the 15-amino acid C-terminal peptide [delta(401-415)], the N-terminal domain exhibits several characteristics of a molten globule state, whereas the C-terminal domain retains native-like, although distinctly different, tertiary structure. Deletion of three C-terminal amino acids [delta(413-415)] also globally affects PGK conformation, although to a much lesser extent. Both C-terminal deletions resulted in a significant decrease in protein stability, as demonstrated by their increased susceptibility to guanidine-induced and thermal denaturation. These results suggest that the formation of a native tertiary fold of PGK requires the presence of a complete polypeptide chain.

The serine receptor of bacterial chemotaxis exhibits half-site saturation for serine binding.

Ligand binding to the serine receptor of Escherichia coli has been studied using isothermal titration calorimetry. Bacterial inner membranes enriched in the serine receptor (Tsr) were titrated as sonicated membrane samples and after solubilization in octyl beta-D-glucopyranoside (OG) to determine the number of moles of ligand bound per mole of receptor (n), the binding constant (Ka), and the enthalpy of binding (delta H) of serine to the receptor. The n value for serine binding to OG-solubilized Tsr protein (n = 0.5) was consistent with one molecule of serine binding to a receptor dimer, but in sonicated inner membrane samples, the n value was smaller (n approximately equal to 0.25), indicating that not all of the binding sites were accessible to added serine. At 7 and 27 degrees C, the values for Ka and delta H were equivalent for the membrane and OG-solubilized samples and were found to be 4.7 x 10(4) M-1 and -15 kcal/mol, and 3.6 x 10(4) M-1 and -18 kcal/mol, respectively. The influence of covalent modification at the sites of methylation on the affinity of the receptor for serine was also investigated, and found to have only a modest effect. The property of half-site saturation is suggestive of models for transmembrane signaling where the receptor subunit interactions are modulated by ligand binding.

Calorimetric studies of serum transferrin and ovotransferrin. Estimates of domain interactions, and study of the kinetic complexities of ferric ion binding.

Human serum transferrin and hen ovotransferrin have been studied by differential scanning calorimetry (DSC), in an effort to quantitatively estimate the free energy of interaction of the N- and C-domains in each protein and to further understand their interaction with chelated ferric ions. In the case of serum transferrin, separate DSC transitions are observed for the two domains while only a single, coupled transition is seen for ovotransferrin. Although domain interactions are somewhat larger for ovotransferrin (-4100 cal/mol) than for serum transferrin (-3100 cal/mol), the major cause of separated transitions for serum transferrin is that the difference in intrinsic folding stability of the N- and C-domains is about 4-fold larger than for ovotransferrin. Chelated ferric ions bind strongly to each site in both proteins and produce changes in Tm by as much as 30 degrees C. When apparent binding constants are estimated from DSC results, these appear to be substantially larger than those estimated previously from equilibrium methods at low temperatures, where very long equilibrium times must be used because of slow ligand release. Although second DSC upscans on each protein show good "reversibility", downscans on serum transferrin revealed that liganded forms of the protein are in fact not in true equilibrium during upscanning, which causes Tm values during upscans to be higher than the true reversible Tm values. The likely reason for this kinetic control over unfolding is the slow release of bound ferric ions and those effects, for technical reasons, cannot be totally eliminated by lowering the scan rate.

Calorimetric studies of the binding of ferric ions to human serum transferrin.

The binding of ferric ions, chelated with nitrilotriacetate, to human serum transferrin (hTF) has been studied using ultrasensitive titration calorimetry. Studies were done in both the presence and the absence of the synergistic bicarbonate anion. It was found that the C-site of hTF is capable of weakly binding bicarbonate (K of 250 M-1, delta H of -8 kcal) at the binding site even before ferric ion is added, although this does not happen to the same extent at the N-site. When preinsertion of the bicarbonate ion occurs, then ferric ion can subsequently bind very quickly to the C-site. Although the chelated ferric ion can bind weakly to the N-site in a fast reaction, the insertion of the bicarbonate ion occurs subsequently in a slow endothermic reaction. Binding of ferric ion to both sites is quickly reversible in the absence of bicarbonate but becomes kinetically controlled for long periods of time once bicarbonate has inserted into the metal-binding site due to the long time required for release of ferric ion. Estimates of the heats of binding to each site, apparent binding constants, and heat capacities of binding are made for different sets of solution conditions. Results from this study are compared to earlier results with ovotransferrin (Lin, L.-N., Mason, A. B., Woodworth, R. C., & Brandts, J. F. (1991) Biochemistry 30, 11660-11669), with major differences and some similarities noted.

Calorimetric studies of the N-terminal half-molecule of transferrin and mutant forms modified near the Fe(3+)-binding site.

The effects of single amino acid substitution on the thermal stability of the N-terminal half-molecule of human transferrin and its iron-binding affinity have been studied by high-sensitivity scanning calorimetry. All site-directed mutations are located on the surface of the binding cleft, and they are D63-->S, D63-->C, G65-->R, H207-->E and K206-->Q. Differential scanning calorimetry results show that the mutations do not significantly alter the conformational stability of the apo-forms of the proteins. The changes in free energy of unfolding relative to the wild-type protein range from 0.83 to -2.4 kJ/mol. The D63-->S, G54-->R and H207-->E mutations slightly destabilize the apo-protein, while the D63-->C and K206-->Q mutations increase its stability by a small amount. However, there are large compensating enthalpy-entropy changes caused by all mutations. All mutants bind ferric ion, but with different affinities. Replacement of Asp-63 by either Ser or Cys decreases the apparent binding constant by 5-6 orders of magnitude. The G65-->R mutation also decreases the apparent binding constant by 5 orders of magnitude. The K206-->Q mutation increases the apparent binding constant by 20-fold, while the H207-->E mutation does not significantly change the apparent iron-binding affinity of the half-molecule.

Calorimetric studies of the binding of ferric ions to ovotransferrin and interactions between binding sites.

Transferrins are two-domain proteins with a very strong site for iron binding located in each domain. Using ultrasensitive titration calorimetry, the binding of ferric ion (chelated with a 2-fold molar excess of nitrilotriacetate) to the two sites of ovotransferrin was studied in detail as well as the binding to the single site in the N- and C-terminal half-molecules. In the presence of excess bicarbonate ion, the binding occurs in two kinetic steps. The fast process of contact binding is instantaneous with respect to instrument response time, is strongly exothermic for the N site and less so for the C site, and corresponds to binding of the chelated ferric ion. The slower process of bicarbonate insertion with concomitant release of nitrilotriacetate occurs on a time scale of 2-20 min over the temperature range 7-37 degrees C and is endothermic for the N site and exothermic for the C site, with rates being significantly slower for insertion at the C site. The delta H of binding is strongly temperature-dependent for both sites, arising from a large negative delta Cp of binding which probably indicates removal of hydrophobic groups from contact with water. When bicarbonate ion is absent, only the fast process of contact binding is seen. Each site within a half-molecule is qualitatively similar to the same site in intact ovotransferrin, although quantitative differences were detected. It was shown that contact binding to ovotransferrin occurs reversibly with free exchange of Fe+3 between N and C sites, while the attachment to either site becomes essentially irreversible after bicarbonate insertion. The strong preference for the first ferric ion to bind to the N site is shown to be due to its larger contact binding constant and the faster rate of bicarbonate insertion, relative to the C site, and is not due to stronger thermodynamic binding after bicarbonate insertion. True equilibrium is achieved only over much longer periods of time. In another series of experiments, direct binding studies were carried out between the two half-molecules under different states of ligation with Fe+3 in the presence of bicarbonate. The results indicate that the two binding sites in ovotransferrin, separated by ca. 40 A, are not independent of one another but communicate as a result of ligand-dependent changes in the heats and free energies of domain-domain interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Study of strong to ultratight protein interactions using differential scanning calorimetry.

Data from differential scanning calorimetry (DSC) may be used to estimate very large binding constants that cannot be conveniently measured by more conventional equilibrium techniques. Thermodynamic models have been formulated to describe interacting systems that involve either one thermal transition (protein-ligand) or two thermal transitions (protein-protein) and either 1:1 or higher binding stoichiometry. Methods are described for obtaining binding constants and heats of binding by two different methods: calculation or simulation fitting of data. Extensive DSC data on 2'CMP binding to RNase are presented and analyzed by the two methods. It is found that the methods agree when binding sites are completely saturated, but substantial errors arise in the calculation method when site saturation is incomplete and the transition of liganded molecules overlaps that of unliganded molecules. This arises primarily from an inability to determine TM (i.e., the temperature where concentrations of folded and unfolded protein are equal) under weak-binding conditions. Results from simulation show that the binding constants and heats of binding from the DSC method agree quantitatively with corresponding estimates obtained from equilibrium methods when extrapolated to the same temperature. It was also found from the DSC data that the binding constant decreases with increasing concentration of ligand, which might arise from nonideality effects associated with dimerization of 2'CMP. Simulations show that the DSC method is capable of estimating binding constants for ultratight interactions up to perhaps 10(40) M-1 or higher, while most equilibrium methods fail well below 10(10) M-1. DSC data from the literature on a number of interacting systems (trypsin-soybean trypsin inhibitor, trypsin-ovomucoid, trypsin-pancreatic trypsin inhibitor, chymotrypsin-subtilisin inhibitor, subtilisin BPN-subtilisin inhibitor, RNase S protein-RNase S peptide, avidin-biotin, ovotransferrin-Fe3+, superoxide dismutase-Zn2+, alkaline phosphatase-Zn2+, and assembly of regulatory and catalytic subunits of aspartate transcarbamoylase) were analyzed by simulation fitting or by calculation. Apparent single-site binding constants ranged from ca. 10(5) to 10(20) M-1, while the interaction constant for assembly of aspartate transcarbamoylase was estimated as 10(37) in molarity units. For most of these systems, the DSC interaction constants compared favorably with other literature estimates, for some it did not for reasons unknown, while for still others this represented the first estimate. Simulations show that for proteins having two binding sites for the same ligand within a single cooperative unit, ligand rearrangement will occur spontaneously during a DSC scan as the transition temperature of the unliganded protein is approached.(ABSTRACT TRUNCATED AT 400 WORDS)

Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: effects on catalysis, activation by sulfate, and thermal stability.

A "hinge-bending" domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Banks et al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183----Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. The Km values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183----Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the Ala----Pro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183----Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, with Tm near 54 degrees C, two transitions are evident for the mutant enzyme with Tm values of about 45 and 54 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple model for proteins with interacting domains. Applications to scanning calorimetry data.

A simple thermodynamic model is formulated for the purpose of interpreting scanning calorimetry data on proteins that have interacting domains. Interactions are quantified by inclusion of an interface free energy, delta GAB, in the thermodynamics of unfolding for multidomain proteins. The assumption is made that delta GAB goes to zero with the unfolding of either domain involved in pairwise interaction, so the interaction term appears to stabilize only the domain with the lower TM. Application of the model to calorimetric data leads to an estimate of -25,000 cal/mol for interactions between the regulatory and catalytic subunits of native aspartate transcarbamoylase and to a value of 0 for delta GAB between the transmembrane and cytoplasmic domains of band 3 of the human erythrocyte membrane. Estimates of changes in delta GAB are also obtained for mutant forms of yeast phosphoglycerate kinase that have been altered in the hinge region between amino-terminal and carboxy-terminal domains. The model is also applied to ligand binding to proteins having domains that communicate through pairwise interaction. It is shown that whenever the delta GAB term is ligand-dependent, then attachment of the ligand to the binding domain will be partially controlled by the other (regulatory) domain. This situation can sometimes be recognized and quantified when calorimetric scans are carried out at varying ligand concentrations. According to the model, the binding of MgATP to the carboxy-terminal domain of phosphoglycerate kinase is strongly stabilized (ca. 20% of the unitary free energy of binding) by participation of the amino-terminal domain, which acts to increase the binding constant 25-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid measurement of binding constants and heats of binding using a new titration calorimeter.

A new titration calorimeter is described and results are presented for the binding of cytidine 2'-monophosphate (2'CMP) to the active site of ribonuclease A. The instrument characteristics include very high sensitivity, rapid calorimetric response, and fast thermal equilibration. Convenient software is available for instrument operation, data collection, data reduction, and deconvolution to obtain least-squares estimates of binding parameters n, delta H degree, delta S degree, and the binding constant K. Sample through-put for the instrument is high, and under favorable conditions binding constants as large as 10(8) M-1 can be measured. The bovine ribonuclease A (RNase)/2'CMP system was studied over a 50-fold range of RNase concentration and at two different temperatures. The binding constants were in the 10(5) to 10(6) M-1 range, depending on conditions, and heats of binding ca. -15,000 cal/mol. Repeat determinations suggested errors of only a few percent in n, delta H degree, and K values over the most favorable concentration range.

Separation of the nativelike intermediate from unfolded forms during refolding of ribonuclease A.

In an effort to determine structural properties of the nativelike intermediate (i.e., IN) which forms during the refolding of RNase A, refolding samples were subjected to rapid HPLC gel filtration which allowed us to separate IN from unfolded forms of RNase. The comparison of these samples, enriched in IN and depleted of unfolded forms, with unseparated control samples at the same stage of refolding allowed certain conclusions to be drawn concerning the properties of IN. First, the results show that the transition from IN to native RNase occurs with only small changes in fluorescence. This means that the major fluorescence changes seen during normal refolding experiments must be associated with changes in proline isomerization of unfolded species and/or with the refolding step itself but not with the IN----N step. Second, the fluorescence assay for isomerization of proline-93 shows that IN exists with proline-93 in a state of isomerization identical with or very similar to native RNase; i.e., proline-93 is cis in IN and not trans as suggested by others. All results are semiquantitatively consistent with our earlier refolding model and not nearly so consistent with alternative models which assume that most or all of the slow-refolding forms of RNase have proline-93 in the incorrect trans state.

Catalysis of proline isomerization during protein-folding reactions.

The enzyme peptidylprolyl cis-trans isomerase (PPI) is known to catalyze proline isomerization in short proline-containing peptides. If PPI can be shown to generally catalyze isomerization of proline residues in proteins, then it would be a valuable diagnostic reagent for recognition of isomerization, which has proven to be extremely difficult to characterize by other methods. In this study, the catalytic effect of PPI on the slow refolding reactions of seven different proteins has been studied, and in only two cases (RNase T1 and cytochrome c) could significant catalysis be seen. PPI also caused no enhancement in the rate for the 'subtle' conformational changes of native concanavalin A or native Fragment I of prothrombin, which have been suggested to be rate-limited by proline isomerization. There was a small effect of PPI observed for the generation of native RNAase A from the fully-reduced form when the glutathione concentration was low. The conclusion from these studies is that PPI can weakly catalyze some protein processes which are rate-limited by proline isomerization, but probably exhibits no measureable catalysis toward others. This somewhat limits the usefulness of PPI as a diagnostic reagent for proline isomerization.

Evidence for the existence of three or more slow phases in the refolding of ribonuclease A and some characteristics of the phases.

The slow refolding kinetics of RNase A have been analyzed, by using a nonlinear least-squares program for deconvoluting the kinetic phases and applying statistical tests for quality of fit. It is found that a minimum of three slow phases are required to fit the kinetic data properly, and this is true whether the method of detection is absorbance of fluorescence. Since the number of phases and the relaxation times for each phase are independent of the method of detection, it is concluded that the same three rate-limiting processes are seen by absorbance and fluorescence. These phases correspond to the XY, CT, and ct phases described in our earlier studies. The fact that fluorescence-detected kinetics are somewhat slower than absorbance-detected kinetics is a trivial effect due not to differences in relaxation times but to the fact that the amplitude of the CT phase is enhanced in fluorescence measurements, at the expense of the faster XY phase, because of intrinsic fluorescence changes associated with the isomerization of proline-93. By use of a new double-jump technique [Schmid, F.X., Grafl, R., Wrba, A., & Beintema, J.J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872], it is shown that proline-93 isomerizes as the rate-limiting step in only one of the three phases, the CT phase, and that this phase involves only 25-30% of the RNase molecules. There is still no indication as to the molecular events that occur in the large, ammonium sulfate dependent XY phase, which is the pathway for formation of the nativelike intermediate.

Refolding of ribonuclease in the presence and absence of ammonium sulfate pulses. Comparison between experiments and simulations.

Experiments have been carried out on ribonuclease A in which refolding in high concentrations of guanidine hydrochloride is either preceded or not preceded by a short ammonium sulfate pulse. Application of the pulse causes the rapid formation of the nativelike intermediate, and the effect of this pulse was determined by using three different methods for monitoring the subsequent refolding reaction: direct absorbance, direct fluorescence, and a double-jump fluorescence unfolding assay which is specific for the isomerization of proline-93. The effect of the pulse is quite different depending on the method of detection. With absorbance detection, the pulse causes a large reduction in the refolding amplitude with no change in the kinetics of the decay curve, while with the fluorescence unfolding assay, the pulse causes no change in the refolding amplitude but produces a large acceleration in the decay kinetics. The results with direct fluorescence are intermediate with some reduction seen in the refolding amplitude and some acceleration in the decay kinetics. The results of these experiments are simulated by using the simple model of Lin and Brandts (1984) [Lin, L.-N., & Brandts, J. F. (1984) Biochemistry 23, 5713] in which proline-93 must be in the correct cis configuration before folding to the native or nativelike state can occur. In all cases, the simulations accurately predict the experimental results for all three methods of detection, without any adjustment of parameter values from those published earlier.(ABSTRACT TRUNCATED AT 250 WORDS)