Changes In Protein Fluorescence Research Articles

Binding of the O-antigen of Shigella dysenteriae type 1 and 26 related synthetic fragments to a monoclonal IgM antibody.

Shigella dysenteriae type 1 possesses an O-antigen whose repeating unit is -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp -(1-->3)-alpha-D- GlcpNAc-(1-->, where Rhap is rhamnopyranosyl, Galp is galactopyranosyl, and Glcp is glucopyranosyl. Using ligand-induced protein fluorescence change, we have measured the affinities of a monoclonal murine IgM for 26 fragments of, or related to, the structure of the O-polysaccharide and of the IgM Fab for the intact O-specific bacterial polysaccharide. Synthetic saccharides used were methyl glycosides to ensure an anomerically defined pyranosyl ring conformation. The galactosyl residue is the only monosaccharide of the antigenic epitope that shows quantifiable binding: approximately 3.0 kcal/mol of binding free energy, depending on the structure and conformation of the fragment it is a part of. Addition of an alpha-(1-->2)-linked rhamnosyl residue increases the free energy of binding significantly. We propose this rhamnopyranosyl-alpha-(1-->2)-galactopyranosyl disaccharide to be the basic determinant of the Shigella O-polysaccharide. Further extension (by linkages as in the natural antigen) of this oligosaccharidic ligand toward the upstream end (in an oligo- (or poly-)saccharide, such as A-->B-->C-->D-->E-->m, where A, B, C, D, and E are sugars and m is any moiety, such as methyl, we define A as the glycosyl- or upstream terminus, and E as the glycoside- or downstream terminus) by rhamnosyl and N-acetylglucosaminyl moieties improves the binding only minimally. The antibody is quite specific for the rhamnosyl-alpha-(1-->2)-galactosyl sequence but less so for the nature of the attachment to the galactosyl residue on the downstream side. Measurements using IgM Fab and the intact O-specific polysaccharide show that the antibody can bind internal segments on the antigen chain. The free energy of binding of this antibody for the disaccharide determinant varies from -delta G of 4.7 to 5.1 kcal/mol, depending on its flanking residues.

Kinetics of conformational changes associated with inhibitor binding to the purified band 3 transporter. Direct observation of allosteric subunit interactions.

Subunit interaction effects were identified for isolated human erythrocyte band 3, the anion exchanger, by observing both static and stopped-flow kinetic protein fluorescence changes associated with inhibitor binding to the intramonomeric stilbenedisulfonate site. We measured the rate of conformational changes associated with reversible binding of H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate). The rate of H2DIDS release was also measured. As a test for subunit interactions, we studied the effect of partial labeling of the band 3 monomer population with H2DIDS on the equilibrium and kinetics of H2DIDS reversible binding to the remaining monomers. The results showed biphasic kinetics for control band 3, with a pseudo-first-order ligand dependence for the fast phase followed by a slow ligand-independent relaxation. A second-order "on" rate constant for the fast phase was determined to be (1.2 +/- 0.1) x 10(7) M-1 s-1, while the associated "off" rate constant was found to be 1.1 +/- 0.5 s-1. From these kinetic constants, we calculated a Kd value of 95 +/- 50 nM, which is in excellent agreement with the Kd value determined at thermodynamic equilibrium (110 +/- 9 nM). Covalent labeling of 75% of the band 3 monomer population with H2DIDS changed the kinetics of the fast phase, slowing the apparent rate by changing the order of the reaction from pseudo-first-order to zero-order. Partial labeling did not affect the ligand-independent relaxation. Separate measurements of the H2DIDS "off" rate also showed a biphasic time course, with a 20-fold difference in apparent rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of modified and unmodified tRNATyr substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

Three species of tRNA(Tyr) have been examined as substrates for the transfer reaction of the tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus: Escherichia coli tRNA(Tyr), B. stearothermophilus tRNA(Tyr) expressed in E. coli, and B. stearothermophilus tRNA(Tyr) that has been transcribed in vitro. The binding of the first two substrates to TyrRS may be readily monitored by stopped-flow studies of tryptophan fluorescence to give the rate and equilibrium constants. The in vitro-transcribed tRNA(Tyr), which lacks the modified bases queuosine and 2-(methylthio)-N6-isopentenyladenosine in the anticodon loop, does not cause a significant change in tryptophan fluorescence upon binding. The three tRNA(Tyr) substrates exhibit very similar steady-state kinetics in the charging reaction. Pre-steady-state kinetics of the transfer reaction, monitored by stopped-flow measurements of the change in protein fluorescence on the addition of tRNA(Tyr) to the E.Tyr-AMP complex, show two exponential changes for the modified tRNA(Tyr) substrates. The first is that due to substrate binding. The second has an identical rate to the single change observed for the reaction with the in vitro-transcribed tRNA(Tyr) and to that monitored by quenched-flow measurements on the formation of Tyr-tRNA(Tyr). Hence, the transfer reaction can be observed by stopped-flow. The dissociation constants (KtRNA) of tRNA from the enzyme and rates of tyrosine transfer (k4) show that all three tRNA molecules are kinetically equivalent substrates for TyrRS. The value of k4 is also similar to that found for authentic tRNA(Tyr) from B. stearothermophilus.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitor binding to the Phe53Trp mutant of HIV-1 protease promotes conformational changes detectable by spectrofluorometry.

HIV-1 protease contains two identical, conformationally mobile loops, known as flaps, which form in part the binding pockets for substrates and inhibitors. We have constructed a site-specific mutant of the protease in which residues Phe-53 and Phe-153 at the end of the flaps have been mutated to Trp residues, in order to incorporate a specific fluorescent probe to monitor conformational changes upon the binding of an inhibitor. The Phe53Trp (F53W) mutant of HIV-1 protease was expressed in Escherichia coli and purified from bacterial lysates. Analysis of the purified mutant protease demonstrated that its kinetic properties were highly similar to those of the wild-type protease. While binding of a potent peptide-analogue inhibitor (Ki = 9 nM) to the wild-type enzyme led to no change in protein fluorescence, a 5-8% increase in fluorescence was observed with the F53W mutant, indicating an enhancement of the Trp fluorescence due to flap movement upon inhibitor binding. Investigation of the kinetics of the F53W protease-inhibitor binding by stopped-flow spectrofluorometry revealed a rapid increase in protein fluorescence upon formation of the enzyme-inhibitor complex. These data were consistent with a one-step mechanistic model of inhibitor binding in which flap movement was concomitant with inhibitor binding, from which respective rate constants of association and dissociation of 2.5 x 10(6) M-1 s-1 and 0.023 s-1 were obtained.

Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.

The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Stopped-flow fluorescence kinetic studies of Glu-plasminogen Conformational changes triggered by AH-site ligand binding

Binding of 6-aminohexanoic acid to the AH-site, a weak lysine binding site in Glu-plasminogen, alters the conformation of the molecule. The kinetics of the binding and the accompanying conformational change are investigated at pH 7.8. 25°C. Changes of intrinsic protein fluorescence were measured as a function of time after rapid mixing in a stopped-flow apparatus. The results reflect a two-step reaction mechanism: Rapid association of Glu-plasminogen and 6-aminohexanoic acid ( K 1 = 44 mM) followed by the conformational change ( k 2 = 69 g −1 and k −2 = 3 g −1) with on overall dissociation constant K 4 = 2.0 mM, Thus the conformational change is rather fast, t 1 2 = 0.01 g. Its importance for the rates or Glu-plasminogen activation reactions is discussed.

Inhibition of Escherichia coli glutamine synthetase by .alpha.- and .gamma.-substituted phosphinothricins

We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.

Postnatal changes in antioxidants, trap and protein fluorescence in healthy preterm infants: Influence of feeding

Postnatal changes in antioxidants, trap and protein fluorescence in healthy preterm infants: Influence of feeding

Urea-induced inactivation, dissociation and unfolding of the allosteric phosphofructokinase from Escherichia coli

The influence of urea on the allosteric phosphofructokinase from Escherichia coli has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, and retention in size-exclusion chromatography. Tetrameric, dimeric, and monomeric forms of the protein can be discriminated by their elution from a high-performance liquid chromatography gel filtration column. Three successive steps can be detected during the urea-induced denaturation of phosphofructokinase: (i) the dissociation of the native tetramer into dimers which abolishes the activity; (ii) the dissociation of dimers into monomers which exposes the unique tryptophan, Trp-311, to the aqueous solvent; (iii) the unfolding of the monomers which disrupts most of the secondary structure. This pathway involves the ordered dissociation of the interfaces between subunits and supports a previous hypothesis (Deville-Bonne et al., 1989). Phosphofructokinase can be quantitatively renatured from urea solutions, provided that precautions are taken to avoid the aggregation of one insoluble monomeric state. The renaturation of phosphofructokinase from urea implies three steps: an initial folding reaction within the monomeric state is followed by two successive association steps. The faster association step restores the native fluorescence, and the slower regenerates the active enzyme. The renaturation and denaturation of phosphofructokinase correspond to the complex pathway: tetramer in equilibrium dimer in equilibrium folded monomer in equilibrium unfolded monomer. It is found that the subunit interface which forms the regulatory site is more stable and associates 40 times more rapidly than the subunit interface which forms the active site.

Carbohydrate-linked asparagine-101 of prothrombin contains a metal ion protected acetylation site. Acetylation of this site causes loss of metal ion induced protein fluorescence change.

Prothrombin fragment 1 (prothrombin residues 1-156) contains two acetylation sites that are protected from derivatization by calcium. The first site was protected by only calcium [Welsch, D. J., & Nelsestuen, G. L. (1988) Biochemistry (second of three papers in this issue)] while the second site was protected by magnesium as well. To identify this second acetylation site, fragment 1 was first acetylated with unlabeled reagent in the presence of magnesium. Metal ions were removed, and the protein was acetylated with radiolabeled reagent. The incorporated radiolabel was stable over long periods of time and at acidic or basic pH as long as elevated temperatures were avoided. The radiolabel was removed by treatment of the protein at pH 10 and 50 degrees C or with 0.2 M hydroxylamine at 50 degrees C for at least 30 min. Proteolytic degradation of the protein showed that the radioactivity appeared in a tryptic peptide corresponding to residues 94-111 of prothrombin. The Lys-97 in this peptide was acetylated but did not contain radiolabel. Amino acid sequence analysis revealed that the radiolabel was associated with an unextracted sequence product. Aglycofragment 1, produced by treatment of fragment 1 with HF, was radiolabeled by this procedure; peptide 94-111 was isolated and was further digested with protease. The major radiolabeled product contained Asn101-Ser102 along with the expected chitobiose attached to Asn-101. NMR analysis revealed the presence of three acetate groups which would correspond to two from the chitobiose plus the incorporated acetate residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein fluorescence changes associated with ATP and adenosine 5'-[gamma-thio]triphosphate binding to skeletal muscle myosin subfragment 1 and actomyosin subfragment 1.

1. The fluorescence changes accompanying the binding of ATP and adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) to myosin subfragment 1 (S1) and actomyosin subfragment 1 (actoS1) have been reinvestigated at 20 degrees C and 1 degree C, pH 7.0, 0.1 M-KCl. 2. Two successive fluorescence enhancements are observed following ATP binding to both S1 and actoS1. 3. The slow fluorescence change has the same rate with S1 and actoS1, and is due to the ATP cleavage step. 4. With actoS1 the fast fluorescence change occurs after dissociation, so a new intermediate, S1 ATP, is required on the actoS1 pathway. 5. The dissociation of actoS1 by ATP gamma S results in a fluorescence enhancement with the same apparent rate as dissociation, but indirect evidence suggests that this too occurs on a dissociated state.

Interaction of spectrin with phospholipids. Quenching of spectrin intrinsic fluorescence by phospholipid suspensions

Phospholipid suspensions prepared of phosphatidylethanolamine, phosphatidylserine and their mixtures are able to influence the intrinsic protein fluorescence of spectrin. In the case of phosphatidylethanolamine suspension up to 75% of protein fluorescence can be quenched. The interaction of phospholipid aggregates with spectrin is modulated by pH and ionic strength. Phospholipids, particularly phosphatidylethanolamine display a ‘stabilizing’ effect against the changes of protein fluorescence induced by increasing ionic strength and by thermal denaturation.

Comparison of the kinetic behavior of human and bovine proteins in the heparin-catalyzed antithrombin III/thrombin reaction

Significant differences between saturation kinetic properties of heparin-stimulated reactions between thrombin and antithrombin III from human and bovine species were observed. In both systems, the apparent K m for antithrombin III was higher than the K D for antithrombin III-heparin interaction, monitored by intrinsic protein fluorescence change. The K m for thrombin and k cat were much higher for proteins of the human species than the bovine species. The apparent K m for one human protein was dependent on the concentration of the other human protein, indicating interaction of the binding events. The reaction product formed from the bovine proteins was a potent inhibitor of the reaction but the product from the human proteins was a poor inhibitor. The major differences between the two species appeared to be related to interaction of thrombin or thrombin derivatives with heparin or heparin-antithrombin III complexes.

Subunit-subunit interactions in TF 1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′- O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF 1 and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant ( K D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF 1 binds 2 mol 1-naphthoyl-ATP per mol enzyme with K D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10 4 M −1s −1. (6) The rate constant for 1-naphthoyl-ATP binding to TF 1 is 6.6 · 10 3 M −1 · s −1 (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase ( k A −1 = 3 · 10 −3 s −1) and a slower second phase ( k A −2 < 0.2 · 10 −3 s −1). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF 1 are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.

Carboxymethylated liver alcohol dehydrogenase: pH dependence of hydride transfer during ethanol oxidation

The rate constant for the hydride transfer step during oxidation of ethanol by cabroxymethylated alcohol dehydrogenase (alcohol:NAD + oxidoreductase, EC 1.1.1.1) is dependent on a group with p K a 7.5. This p K a is higher than that for the native enzyme. A mechanism is proposed to account for the ionisation and the protein fluorescence change which occur during formation of the carboxymethylated enzyme-NAD +-ethanol complex.

Effect of the lyotropic series of anions on denaturation and renaturation of 20β-hydroxysteroid dehydrogenase

The effect of the lyotropic series of anions on the stability and renaturation of tetrameric 20β-hydroxysteroid dehydrogenase (17,20β,21-trihydroxysteroid:NAD + oxidoreductase, EC 1.1.1.53) was investigated. The variations in enzymatic activity were correlated with the changes in protein fluorescence, circular dichroism, reactivity of histidine residues and molecular weight. High concentrations of salting-out anions (phosphate, citrate, sulphate) were found to stabilize the enzyme markedly and increase the renaturation yield of the urea-denaturated enzyme. Phosphate, for instance, induced the highest stabilization at about 1.2 M and the maximum reactivation (66%) at 0.5 M. At low anion concentration (0.01 M), the reactivation was only 7%. The renaturation property of salting-out anions seems to be due to their stabilizing effect on the end-product, i.e., the assembled tetramer. Salting-in anions (perchlorate, thiocyanate, iodide) inactivated the enzyme. At moderate anion concentrations (no greater than 0.25 M) the inactivation, which occurred slowly, without tetramer dissociation and with minor modifications of enzyme conformation, was fully reversed by concentrated phosphate or by saturating concentrations of NADH. In contrast, the inactivation induced by high anion concentrations (1–2 M) was rapid, irreversible and linked to considerable modifications of enzyme conformation.

The interactions of Rose Bengal and other aromatic anionic dyes with mannitol-1-phosphate dehydrogenase fromEscherichia coli

The interaction of Rose Bengal with mannitol-1-phosphate dehydrogenase has been investigated. Binding of this aromatic anionic dye causes a quenching of the protein fluorescence and various changes in the spectral properties of the dye. As is the case with other dehydrogenases, the titration of the enzyme with Rose Bengal, monitoring enhancement in the dye fluorescence at 590 nm, or quenching of the protein fluorescence, can be described by a simple binding model: one dye binding site per enzyme subunit with a dissociation constant of ∼2 µM. However, kinetic studies indicate a more complex scheme, since Rose Bengal induces a biphasic time-dependent inhibition of the enzyme. The first phase is over in 1–5 min and is partially reversible, while the second phase is essentially irreversible and continues beyond 1 h. The dyes 8-anilino-1-naphthalene sulfonate and 2-p-toluidinylnaphthalene-6-sulfonate also cause biphasic time-dependent inhibitions of the enzyme. Only mannitol-1-phosphate, and fructose-6-phosphate in the presence of NAD+, show high levels of protection against these inhibitory processes. The different effects of coenzymes and substrates on the dye-induced inhibitions support earlier observations from fluorescence studies (preceding paper). A binding scheme describing the interactions of Rose Bengal with the enzyme that is consistent with the experimental results is presented.

The refolding of denatured rabbit muscle creatine kinase. Search for intermediates in the refolding process and effect of modification at the reactive thiol group on refolding.

A number of aspects of the refolding of denatured rabbit muscle creatine kinase have been studied. Addition of substrates has no effect on the rate or extent of regain of activity. The changes in protein fluorescence during refolding broadly parallel the regain of activity. A study of the susceptibility of the enzyme to proteolysis during refolding indicates that there is no significant accumulation of folded, but inactive, intermediates in the folding process. Modification of the reactive thiol group on each subunit of the enzyme by small reagents such as iodoacetate or iodoacetamide prior to denaturation has only a small effect on the rate of subsequent refolding. However, modification by the bulky reagent 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate has a very large effect on the ability of the enzyme to refold after denaturation.

Calcium binding to α-lactalbumin: Structural rearrangement and association constant evaluation by means of intrinsic protein fluorescence changes

Calcium binding to alpha-lactalbumin : structural rearrangement and association constant evaluation by means of intrinsic protein fluorescence changes.