Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions

The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([K_Q]eff) and the fraction of fluorescence available ([f_a]eff) to the quencher are also calculated. In contrast to its effect on tryptophan, CH₃COO^? quenches tyrosyl fluorescence and ClO₄^? does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan-containing proteins. (1) NO₃^?, NO₂^?, Br^?, and I^? have high [K_Q]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO₄^? and SCN^? also quench fluorescence of tryptophan in proteins and have lower ([K_Q]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [(f_a)eff] = 1. (3) Cl^?, CH₃COO^?, and SO₄? do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO₄⁼, CH₃COO^? ? Cl^? < ClO₄^? < SCN^? < Br^? < I^? < NO₃^? < NO₂^?.     

The solution behavior of poly-L-proline: II. Fluorescence behavior of labeled polyproline in water and conentrated aqueous neutral salt solytions

Fluorescence depolarization experiments performed on labaled poly-L -proline Forme II suggest the occurrence of aggrgation in water while 6M guanidinium-HCl induces dissociation. The solvent 4M CaCl₂ results in a reduction of polymer structural orgganization. These findings corroborate suggestion of polyproline aggregation and solution behavior in aqueous neutral salt solytion (see preceding article).     

Stability and thermodynamics of polyuridylic acid-deoxyadenosine complexes in aqueous neutral salt solutions

Using the thermodynamic analysis and methodology of Hill (Biopolymers, 12 , 257 (1973)) for the treatment of optical thermal transition data the effects of various neutral salt additives on the stability and thermodynamics of the poly U–deoxyadenosine interactions that lead to the formation of triple-stranded helical polymer–monomer complexes have been studied. In order of increasing molar effectiveness as polyU–deoxyadenosine complex stability perturbants (pH 7 and in the presence of 1 M NaCl), the various ions may be ranked: SO₄^?2 < Cl^? < Br^? < ClO₄^?; and (CH₃)₄ N⁺ < Li⁺ < Rb⁺ ～ Na⁺ < K⁺ < (CH₃ CH₂)₄ N⁺ < urea < Guan⁺ ～ (CH₃(CH₂)₂)₄ N⁺. Destabilizing neutral salt additives (e.g., NaClO₄) caused a decrease in the absolute magnitude of the apparent enthalpy and entropy of binding relative to the values determined in the presence of NaCl. By contrast, stabilizing additives (e.g., Na₂ SO₄) had the opposite effect on these parameters. Along a melting curve the apparent differential heat of complex formation calculated for the binding of deoxyadenosine to poly U in 1 M NaCl appeared to vary linearly with θ, the extent of fractional binding. For such a linear dependence it can be shown that the integral heat (usually determined calorimetrically) equals the differential heat at θ = 0.5. Correcting the apparent differential heat calculated at θ = 0.5 for ligand activity resulted in values for the integral heat of binding of deoxyadenosine to poly U in 1 M NaCl of ?13 to ?16 kcal/mol. Binding isotherms determined in the presence of different inorganic electrolytes could be superimposed provided that different temperatures were compared. However, the additive (CH₃)₄NCl, which has been shown to interact preferentially with A-T rich regions of DNA (Shapiro, Stannard, and Felsenfeld, Biochemistry, 8 , 3233 (1969)) resulted in a considerably broadened binding isotherm indicating less cooperativity.     

Volatile transport in frozen aqueous solutions. II. Influence of system parameters

Frozen aqueous butanol solutions are evaluated for the influence of sample preparation and equilibration conditions on the loss of butanol.It is shown that an increase of equilibration temperature results in increased butanol loss, both water independent and water dependent. The freezing rate will influence the equilibration retention level, with faster freezing resulting in smaller butanol loss. An increase in butanol concentration gave the same percentage of butanol loss, i.e., the amount of butanol lost was directly proportional to the initial concentration.     

Diffusion of lysozyme chloride in water and aqueous potassium chloride solutions.

The diffusion of lysozyme chloride in aqueous solution has been studied at 25 degrees C using the Goüy interferometric technique. The concentration dependence of the diffusion coefficient in water has been measured over the concentration range 1.1599-9.1556 gcm-3 and the results suggest a value of D 25, w at infinite dilution of 5.838 x 10(-6) cm2s-1. The variation in diffusion coefficient with ionic strength has also been considered by following the diffusion of 0.45% lysozyme chloride in a series of potassium chloride solutions. The value of D in 0.15 M KCl has been found to be approximately one quarter of that in water alone an the diffusion coefficient has been shown to increase markedly as the KCl concentration is reduced below 0.05 M. Interpretation of these observations involves consideration of solution electrostatic effects.     

Pressure-temperature stability of DNA in neutral salt solutions

The effect of pressure on the melting temperature of DNA (Cl. perfringens) has been examined in several concentrations of neutral salt by measurement of uv absorbance. The results indicate that the apparent transition volume increases as the salt concentration, and hence the melting temperature, is raised, and suggest that the transition occurs without a change in volume at a T_m of 59°C. Additional experiments were conducted in an effort to determine whether transition behaviour can be found in other regions of pressure and temperature; no additional transition behaviour was observed in experiments conducted with temperatures as low as ?21.85°C at 2000 atm and pressures in excess of 9000 atm at 58°C.     

The solution behavior of poly-L-proline: I. Light scattering studies in water and concentrated aqueous neutral salt solutions

The solution bebavior of poly-L -proline Form II has been studied in water and aqueous salt solutions by both elastic and quasi-elastic light -scattering techniques. The results of this study suggest that polyproline Form II can exist in water at 24 °C as an associated polymer complex and that certain salts which do not appear to affect the helix integrity, e.g., guanidinium-HCl, resutl in dissociation of the aggregate. Other neutral salts, of the variety effective in mediating unfolding of the Form II helix (e.g., 4M NaClO₄) also induce aggregate dissociation, but 4M CaCl₂ results in enhanced aggregation of polyproline. Kinetic experiments indicate that a time of 20 hours is necessary for the completion of the “large” to “small” transformation (at 22°C) which is induced by the addition of 4M NaClO₄. Thus it appears that neutral salts additives in aqueous solutions of polyproline influence both the state of aggregation and the conformation of this polymer.     

Dissociation and isolation of chromatin proteins in salt solutions by an aqueous two-phase system

An aqueous two-phase system containing 7% Dextran T 500-5% polyethylene glycol (PEG) 6000 has been adopted for rapid selective stepwise extractions of high-mobility-group proteins and histones from both isolated chromatin and intact nuclei of calf thymus. After the dissociated proteins in the PEG phase were precipitated with 20% trichloroacetic acid at 4 degrees C, proteins were recovered from this phase by solubilization of PEG with acidified acetone at room temperature. This method allows preparation of nuclei depleted of histone H1.     

Properties of monovalent nickel complexes with tetraaza-macrocyclic ligands in aqueous solutions

The effects of N-alkylation on the redox potential of the couples NiLⁱ²⁺/NiLⁱ⁺, L = tetraaza-14-membered-macrocyclic ligands, and on the properties of the monovalent nickel complexes in aqueous solutions are reported for 14 complexes. The spectra and lifetimes of the NiLⁱ⁺ complexes are reported. The self-exchange rates for the couples NiLⁱ²⁺/NiLⁱ⁺ were determined. Two of the ligands were synthesized for the first time for this study. Cyclic voltammetry and pulse radiolysis were used. The results point out that: (i) N-alkylation always shifts the redox potential to a less cathodic one; this effect stems to a large degree from the decrease in the solvation energy of the complex caused by the N-alkylation of the ligand. (ii). The lifetime of the monovalent complexes is not linearly related to the redox potential of the NiLⁱ²⁺/NiLⁱ⁺ couples. (iii) The NiLⁱ⁺ complexes exist in several isomeric forms; the rate of the isomerization depends on the structure of the ligand. (iv) Different isomers of NiLⁱ⁺ may be formed when the complex NiLⁱ²⁺ is reduced by different reagents; therefore, the pulse-radiolytically formed NiLⁱ⁺ complexes might have different properties than those formed electrochemically.     

The DNA melting transition in aqueous magnesium salt solutions.

The melting transition of the magnesium salt of DNA has been systematically examined in the presence of various types of anions. The addition of ClO4- to a concentration of 3.0 N results in the biphasic optical transition, with the first phase exhibiting rapid reversibility and independence of the DNA concentration. This subtransition, which is interpreted as an intramolecular condensation to a collapsed form of DNA, is followed by a DNA concentration-dependent aggregation reaction. The aggregation can be reversed by increasing the ClO4- concentration to 6.0 N while elevating the temperature to post-transition levels. Alternatively, both the collapse and the aggregation can be prevented by melting in the presence of trichloroacetate, the most strongly chaotropic solvent for DNA which has been reported (K. Hamaguchi and E. P. Geiduschek (1962), J. Am. Chem. Soc. 84, 1329). The forces responsible for mediating both the collapse and the aggregation are superficially similar to those involved in maintaining duplex stability. The collapsed form, in particular, possibly possesses features in common with the condensed structures which can be produced in aqueous solution of certain polymers, such as polyethylene glycol (Lerman, L.S. (1971), Proc. Natl. Acad. Sci. U.S.A. 68, 1886).     

Thermally induced conformational transition of double-stranded xanthan in aqueous salt solutions.

The thermally induced conformational transition of double-stranded xanthans (degree of pyruvate substitution, DSp = 0.45) having Mw = 3.1, 5.7, and 20.3 x 10(5) has been studied in aqueous salt solutions by high-sensitivity differential scanning calorimetry (DSC). The double strandedness of these samples in the ordered conformation was ascertained by the value of mass per unit length, ML = 2090 +/- 270 g mol-1 nm-1, which was determined from the contour length obtained by electron microscopic observations and the molecular weight by light scattering measurements. The temperature at half completion of the transition T 1/2 for these samples increased linearly with the logarithm of the cation (Na+, K+) concentration. The plot of 1/T1/2 vs the natural logarithm of cation (Na+) concentration in mM for the sample with Mw = 5.7 x 10(5) (15-SX) yielded the equation 10(3)/T1/2 = 3.45-0.159 ln [Na+]. The specific enthalpy delta hcal for 15-SX, essentially independent of salt concentration above 20 mM, was 8.31 +/- 0.39 J/g (SD, n = 6). No systematic dependence of molecular weight on the transition temperature and the enthalpy was observed. Application of the Manning polyelectrolyte theory to the system using the DSC data suggested that the separation of the double strand of xanthan into two single chains was not completed at the temperature where the endothermic peak was finished. This suggestion is consistent with recent findings by light scattering measurements as a function of temperature. Our DSC study was extended to include four other samples from various sources. It was found that T1/2 and delta hcal depend on the pyruvate contents of the samples. For example, the t1/2 (t1/2/degrees C = T1/2/K - 237.15) values for samples with high pyruvate content (DSp = 0.9) and depyruvated (DSp = 0.14) in 20 mM aqueous NaCl were 48.8 and 85.3 degrees C, respectively. Two other samples showed relatively broad DSC curves having shoulders, which were resolved into two independent components. Thermodynamic parameters for each component were examined as a function of salt concentration, and the results obtained were interpreted in terms of the heterogeneity of the pyruvate content of the samples.     

Interaction of phosphate with monovalent cation uptake in yeast.

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb+ uptake shows saturation kinetics and the phosphate concentration at which half-maximal inhibition is observed is equal to the Km of phosphate for the sodium-independent phosphate uptake mechanism. The kinetic coefficients of Rb+ and TI+ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased. In the case of Na+ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na+ uptake via a sodium-phosphate cotransport mechanism. Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimethylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium uptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.     

Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts

We have reported [Saito, M., Saito, M., & Rosenberg, A. (1984) Biochemistry 23, 1043-1046] that the monovalent cationic ionophore monensin reduced the incorporation of labeled galactose into oligosaccharidyl glycosphingolipids (globotriaosylceramide, globotetraosylceramide, and gangliosides) and induced a cellular accumulation of glucosyl- and lactosylceramide in cultured diploid human fibroblasts. We have undertaken further studies on the effects of monensin and made comparison with the effects of related monovalent cation transporters on plasma membrane glycosphingolipid anabolism in human fibroblasts. Our results demonstrate that ionic flux can markedly influence glycosphingolipid synthesis, and they indicate that, like glycoprotein, the sites of glycosylation of the initial, precursor glycosphingolipids are different from the sites of higher glycosylation. At a concentration of 10(-7) M, monensin induced the maximum inhibition of incorporation of labeled galactose into polyglycosyl sphingolipids: globotriaosylceramide, globotetraosylceramide, and gangliosides; increased incorporation of labeled galactose into glucosyl- and lactosylceramide was clearly evident, and their content rose measurably in the cell at concentrations of monensin as low as 10(-8) M. These effects of monensin were reversible. Incorporation of labeled galactose into higher glycosylated neutral glycosphingolipids and gangliosides slowly resumed, and the accumulated glycosylceramide diminished after removal of monensin from the culture medium. Ouabain (plasma membrane Na+,K+-ATPase inhibitor) and A23187 (Ca2+ ionophore) also caused a rapid increase in incorporation of labeled hexose into glucosylceramide and decreased its incorporation into higher neutral glycosphingolipids and into gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)