pH-induced transition and Zn2+-binding properties of bovine prolactin.

A pH-induced conformational transition was found in bovine prolactin within the physiologically significant pH region from 6.5 to 8.5. The thermal stability of prolactin at pH 6.5 is essentially higher than at pH 8.5. Bovine prolactin binds zinc ions with an apparent association constant of 2 x 10(5) M(-1) at pH 6.5 and 1 x 10(4) M(-1) at pH 8.5. The pH dependence of both thermal stability and zinc binding surrounding the pKa of histidine suggests that these residues plays a key role in the structural integrity of bovine prolactin.

Pb2+ and Hg2+ binding to alpha-lactalbumin.

Interactions of human alpha-lactalbumin with Pb2+ and Hg2+ were studied by intrinsic protein fluorescence. Lead ions bind to the strong Ca2+ binding site of alpha-lactalbumin (association constant Kass approximately 2 x 10(6) M-1) with concomitant spectral changes which are similar to those induced by the binding of Ca2+. Pb2+ also binds to the strong Zn2+ site with Kass approximately 10(5) M-1 and some secondary binding site(s) (which probably contain histidine residues) with apparent Kass approximately 10(4) M-1, causing pronounced aggregation of the protein. Mercury ions bind to alpha-lactalbumin at the primary Zn2+ sites with Kass approximately (1-4) x 10(4) M-1, although the stoichiometry of the binding depends on the conformational state of the protein. Secondary Hg2+ binding sites were suggested to contain histidines, while the strong Hg2+ site contains carboxylates in the coordination sphere and seems to coincide with the strong Zn2+ site. The binding of both Pb2+ and Hg2+ decreases the thermal stability of the Ca(2+)-loaded protein and in some conditions causes pronounced protein aggregation.

Interactions of alpha-lactalbumins with lipid vesicles studied by tryptophan fluorescence.

Complexes of alpha-lactalbumin (alpha-LA)1 with dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) liposomes at pH 8 and at pH 2 have been obtained by means of gel filtration. Thermal denaturation of alpha-LA complexes of DMPC or DPPC at pH 8 was found to depend on the saturation of protein by metal cations. The intrinsic fluorescence of DMPC-alpha-LA and DPPC-alpha-LA was sensitive to two thermal transitions. The first transition corresponded to the Tc of the lipid vesicles, while the second transition arose from the denaturation of the protein. Fluorescence spectrum position suggested that at low temperature tryptophan accessibility increases upon protein-DMPC or protein-DPPC association. At temperatures above the protein transition (70 degrees C) tryptophan appears to interact significantly with the apolar phase of DMPC and DPPC, evidenced by spectral blue shifts. Whereas the free protein at pH 2 adopts the molten globule (MG) state and is characterized by the absence of a thermal transition, the rapidly-isolated DMPC-alpha-LA complex was characterized by the appearance of a distinct fluorescence thermal transition between 50 and 60 degrees C. This result is consistent with a model of a partially-inserted form of alpha-LA which may possess some degree of tertiary structure and therefore unfolds cooperatively.

[The effect of phalloidin on stability of F- and G-actin]. / Vliianie falloidina na stabil'nost' F- i G-aktina.

Intrinsic tryptophan fluorescence and fluorescence of a hydrophobic probe bis-ANS were used to study the effect of phalloidin, a bicyclic heptapeptide toxin, on stability of monomeric (G) and polymeric (F) actin. It was found that bis-ANS fluorescence is sensitive to the actin polymerization process. Phalloidin in concentrations from 1 to 2 molecules per 1 actin molecule shifts the thermally induced unfolding transition in F-actin toward about 15 degrees C higher temperatures. The stabilizing effect of phalloidin is even more evident in the case of urea denaturation of F-actin. Moreover, phalloidin stabilizes against denaturing by not only F-actin, but G-actin as well, showing direct stabilizing interactions between phalloidin and G-actin.

Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.

Binding of Zn(II) ions to alpha-lactalbumin.

The binding of Zn(II) ions to human and bovine alpha-lactalbumin has been studied by fluorescence, scanning microcalorimetry, and proteolytic digestion. The intrinsic tryptophan fluorescence spectrum of Ca(II)-loaded alpha-lactalbumin is insensitive to Zn(II) binding to the strong cation binding sites (Zn:protein ratios up to 20), yet the thermal denaturation transition, as detected by intrinsic fluorescence, is shifted toward lower temperatures. On the other hand, low concentrations of Zn(II) ([Zn]:[protein] less than 1) shift heat sorption curves toward lower temperatures. It was concluded that alpha-lactalbumin possess several relatively strong Zn(II) binding sites, which are filled sequentially, the process being accompanied by protein aggregation. The strongest Zn(II) binding (5 x 10(5) M-1) increases its susceptibility to tryptic and chymotryptic digestion, slightly decreases its affinity for the fluorescent probe, bis-ANS, and alters its interactions with UDP-galactose. Zn(II) binding to aggregated forms of alpha-lactalbumin increases its affinity to bis-ANS.

Calcium-regulated interactions of human alpha-lactalbumin with bee venom melittin.

Affinity chromatography, fluorescence and circular dichroism spectroscopy methods have been used to study the interaction of melittin, a 26-residue peptide from bee venom, with Ca2(+)-binding alpha-lactalbumin from human milk. It has been revealed that melittin binds to the apo- and acidic states of alpha-lactalbumin while the presence of Ca2+ makes the interaction essentially weaker. The association constant for the complex of melittin with apo-alpha-lactalbumin determined from spectropolarimetric melittin-titration data is 2 X 10(7) M-1. The complexation of alpha-lactalbumin with melittin decreases its affinity to Ca2+ by three orders of magnitude. The interaction of apo-alpha-lactalbumin with melittin causes some changes in the environment of its aromatic amino acid residues and drastically alters the conformation of melittin, increasing its alpha-helical content but leaving its single tryptophan residue accessible to water. In the case of the acidic state of alpha-lactalbumin the interaction does not induce an increase in alpha-helical content of melittin.

Interactions of parvalbumins with model phospholipid vesicles.

Interactions of Ca2+-binding proteins, parvalbumins, with model vesicles formed with both synthetic (dipalmitoylphosphatidylcholine) and natural (phosphatidylcholine and phosphatidylethanolamine) phospholipids have been revealed and studied by means of gel-chromatography, electron microscopy, intrinsic fluorescence and microcalorimetry methods. There are at least two populations of liposome-bound parvalbumin one of which has higher affinity to the liposomes (effective binding constant about 10(6) M-1) than the other one. The interaction is modulated by Ca2+ and Mg2+ ions and induces changes in properties of both parvalbumin and liposomes.

Interaction of alpha-lactalbumin with Cu2+.

It has been shown by intrinsic fluorescence spectroscopy that alpha-lactalbumin has several Cu2+ -binding sites per molecule. The Ca2+ -loaded protein binds two or more Cu2+ per molecule with an association constant of about 3 X 10(3) M-1. Apo-alpha-lactalbumin binds one Cu2+ per molecule with association constant 8 X 10(4) M-1 and from two to three Cu2+ with an association constant of about 4 X 10(3) M-1. The results obtained from spectrofluorometric pH titration of alpha-lactalbumin in the acidic pH region show the possible involvement of histidine residues in the coordination of Cu2+. The binding of Cu2+ to alpha-lactalbumin lowers significantly its thermostability and stability towards urea denaturation. The stability of Cu2+, Ca2+-alpha-lactalbumin against thermal and urea denaturation is similar to that of the apo protein. The thermal transition in Cu2+, Ca2+-alpha-lactalbumin occurs within the region of physiological temperatures which may suggest the existence of some thermal regulation of its functioning in vivo.

Environment of tryptophan residues in various conformational states of alpha-lactalbumin studied by time-resolved and steady-state fluorescence spectroscopy.

Decay curves for tryptophan fluorescence of bovine and human alpha-lactalbumin in different states (metal-free and Ca2+ or Mg2+-loaded states of the native and thermally denatured proteins) have been measured at different wavelengths. The curves are best fitted by a sum of three exponents assigned to emission of individual tryptophan residues. The results suggests that the red shift of the fluorescence spectrum of alpha-lactalbumin caused by release of the bound Ca2+ or thermal denaturation is due to changes in the environment of all emitting tryptophan residues.

[Kinetics of dissociation of alpha-lactalbumin complexes with Ca2+ and Mg2+ ions]. / Kinetika dissotsiatsii kompleksov alpha-laktal'bumina s ionami Ca2+ i Mg2+.

Kinetics of dissociation of the complexes of bovine alpha-lactalbumin with Ca2+ and Mg2+ ions induced by mixing of the Ca2+- or Mg2+-loaded protein with the chelator of divalent cations EDTA has been studied by means of intrinsic fluorescence stopped flow method. Within the temperature region from 10 to approximately 37 degrees C the fluorescence kinetics curves for the Ca2+ removal are well fitted by one exponent with the rate constant ranging from 6.10(-3) to 1 s-1. Taking into account rather low rate of the fluorescence changes, one can assume that the limiting stage in this case is the dissociation of the single bound Ca2+ ion from the protein but not a conformational change which occurs after the Ca2+ dissociation. At temperatures above 37 degrees C the kinetics curves are best fitted by two exponents. The second exponent seems to be due to the denaturation of the apo-form of alpha-lactalbumin which takes place at these temperatures. The values of the dissociation rate constants of Mg2+ practically coincide with the values for Ca2+.

[The effect of calcium and magnesium ions on the interaction of calcium-binding proteins parvalbumin and alpha-lactalbumin with dipalmitoylphosphatidylcholine vesicles]. / Vliianie ionov kal'tsiia i magniia na vzaimodeistvie kal'tsiisviazyvaiushchikh belkov parval'bumina i alpha-laktal'bumina s vezikulami dipal'mitoilfosfatidilkholina.

Interactions of the calcium binding proteins, like parvalbumins pI 4.2 and p15.0 and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles have been studied by means of scanning microcalorimetry and intrinsic tryptophan, tyrosine and phenylalanine fluorescence. The interactions are modulated by the Ca2+ and Mg2+ binding to the proteins and induce some changes in the physical properties of both the proteins and the liposomes. The liposomes increase the thermal stability of the Mg2+-loaded and metal-free parvalbumin. Ca2+-loaded alpha-lactalbumin interacts with the liposomes in its native state, while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The interactions of both proteins with the liposomes affect the phase transition from gel to liquid-crystalline state in the liposomes. The results of the microcalorimetric and spectrofluorometric studies are corroborated by the data obtained by means of gel-chromatography on Sepharose 4B.

Interactions of calcium binding proteins, parvalbumin and alpha-lactalbumin, with dipalmitoylphosphatidylcholine vesicles.

Interactions of Ca2+ binding proteins, pike (Esox lucius) parvalbumins pI 4.2 and 5.0, and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles were studied by means of scanning microcalorimetry and intrinsic tyrosine and tryptophan fluorescence methods. The interactions of pike parvalbumins are modulated by Ca2+ and Mg2+ binding to the protein and induce some changes in the physical properties of both the proteins and liposomes. Liposomes increased thermal stability of Ca2+-loaded parvalbumin and decreased thermal stability of both Mg2+-loaded and metal-free protein. The interaction of parvalbumin with liposomes affects the phase transition from gel to liquid-crystalline state in liposomes. Ca2+-loaded alpha-lactalbumin interacts with liposomes in its native state while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The results of the microcalorimetric and spectrofluorometric studies are supported by data obtained by means of gel-chromatography on Sepharose 4B. It may be suggested that these metal-modulated interactions of Ca2+-binding proteins with membranes have some functional significance.

Stopped-flow kinetic studies of Ca(II) and Mg(II) dissociation in cod parvalbumin and bovine alpha-lactalbumin.

The dissociation kinetics of complexes of bovine alpha-lactalbumin and cod parvalbumin with Ca(II) and Mg(II) ions induced by mixing of a Ca(II)- or MG(II)-loaded protein with a chelator of divalent cations (EDTA or EGTA) have been studied by means of the stopped-flow method with intrinsic protein fluorescence registration. Within the temperature interval from 10 to approx. 37 degrees C kinetic curves for Ca(II) removal from alpha-lactalbumin are monoexponential with a rate constant ranging from 0.006 to 1 s. Taking into account the rather low rate of fluorescence changes, one can assume that the limiting stage in this case is the dissociation of the single bound Ca(II) ion from the protein and not a conformational transition which occurs after Ca(II) dissociation. At temperatures above 37 degrees C the kinetic curves require at least two exponential terms for a satisfactory fit. The second exponential seems to be due to denaturation of the apo form of alpha-lactalbumin which takes place at these temperatures. The values of the dissociation rate constants for Mg(II) bound to alpha-lactalbumin practically coincide with those for Ca(II). Within the temperature interval 10-30 degrees C the kinetic curves for Ca(II) and Mg(II) removal from parvalbumin are best fitted by a sum of two exponential terms identified as arising from the dissociation of cations from the two binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

[Kinetics of dissociation of parvalbumin complexes with calcium and magnesium ions]. / Kinetika dissotsiatsii kompleksov parval'bumina s ionami kal'tsiia i magniia.

Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit.

Effects of cation binding on the thermal transitions in calmodulin.

The thermal transitions in different forms of bovine brain calmodulin (0, 1, 2, 3 and 4 bound Ca2+ ions per molecule) have been studied by means of microcalorimetry, intrinsic tyrosine fluorescence, circular dichroism and infrared spectroscopy. The heating of the apoprotein from 5 to 110 degrees C induces at least three unfolding transitions. The heating of Ca2+-loaded calmodulin causes at least two structural transitions, one of which occurs at relatively low temperatures, from approx. 30 to approx 50 degrees C. The binding of the biologically significant Ca2+, Mg2+, Na+ and K+ ions has been measured at 12, 20, 28, 37 and 50 degrees C by means of the fluorescence method. The values of the binding parameters for these cations do not depend on temperature within the range 12 to 50 degrees C. It has been proposed that the temperature independence of the metal-ion-binding properties of calmodulin is achieved due to the temperature-induced structural changes, which adjust the protein conformation in such a way that the protein-binding parameters remain constant.

Sodium and potassium binding to parvalbumins measured by means of intrinsic protein fluorescence.

The binding of Na+ and K+ to whiting parvalbumin (pI 4.4) and pike parvalbumins (pI 4.2 and 5.0) results in a shift of the tryptophan fluorescence spectrum towards shorter wavelengths by 2-4 nm for the whiting protein and in a rise of the tyrosine and phenylalanine fluorescence quantum yield for the pike proteins. The effective binding constants of Na+ and K+ to parvalbumins are within the range of 10 M-1 to 100 M-1. Physiological concentrations of Na+ and K+ lower the affinity of whiting parvalbumin for Ca2+ and Mg2+ by almost an order of magnitude.

Comparative study of physiochemical properties of two pike parvalbumins by means of their intrinsic tyrosyl and phenylalanyl fluorescence.

Physicochemical properties of two pike parvalbumins (pI 5.0 and 4.2), belonging to two different gene lineages, have been studied by their intrinsic tyrosine and phenylalanine fluorescence. The CD sites of these paravalbumins have similar affinities to Ca2+ (and Mg2+) ions, but the EF sites of the proteins have very different affinities to these ions. This results in differing stabilities of these parvalbumins to pH-, urea-, and temperature-induced denaturation. The structure of pike parvalbumin pI 5.0, which binds Ca2+, and Mg2+ ions more tightly, is more stable than that of parvalbumin pI 4.2. Both proteins have higher affinities for Na+ ions than for K+ ions.

[Binding of Mg ions with alpha-lactalbumin studied by fluorescent spectroscopy]. / Issledovanie sviazyvaniia ionov Mg al'fa-latal'buminom metodom fluorestsentnoi spektroskopii.

Titration of metal-freed bovine alpha-lactalbumin with Mg2+ ions causes a two-stepped decrease in the fluorescence quantum yield and a pronounced spectral shift towards shorter wavelengths. It seems to reflect conformational changes induced by the binding of two Mg2+ ions to the protein molecule which results in a transfer of some tryptophan residues from the protein surface into an interior part of the protein in rigid unpolar environment. The Mg2+ association constants evaluated from the fluorimetric Mg2+-titration are 2x10(3) and 2x10(2) M-1. Mg2+ ions in millimolar concentrations almost do not influence the binding of Ca2+ ions to protein. It is assumed that Ca2+ and Mg2+ ions are bound to different sites on alpha-lactalbumin. Mono-calcium, mono-magnesium, bi-magnesium and apo-forms of alpha-lactalbumin are distinct in their fluorescence properties which suggests the difference in the conformation of these forms. The binding of Ca2+ and Mg2+ ions to alpha-lactalbumin has to modulate its function.

[Binding of Ca2+ ions to bovine alpha-lactalbumin. Intrinsic protein fluorescence changes]. / Sviazyvanie ionov Ca2+ al'fa-laktal'buminom iz korov'ego moloka. Issledovanie po izmeneniiam sobstvennoi belkovoi fluorestsentsii.

Binding of Ca2+ ion to bovine alpha-lactalbumin molecule causes a conformational change reflected in an almost two-fold decrease of the fluorescence quantum yield and a rather pronounced spectral shift towards shorter wavelengths (by ca. 20 nm). The changes could be interpreted as a result of a transfer of highly exposed tryptophan residue(s) into a rigid internal part of the protein molecule containing effective quenching groups (probably, vicinal disulphide bridges). The Ca2+ binding constant evaluated from EGTA--and pH-titrations is (3-6) X 10(8) M-1. The results of the spectrofluorometric pH-titration of alpha-lactalbumin in the presence of various Ca2+ concentrations suggest that the well-known acid conformational change in alpha-lactalbumin is due in fact to a competitive replacement of the bound Ca2+ by three H+ ions (pK = 5.0 +/- 0.1) in the Ca2+ binding site.