Spectrofluorimetric determination of nickel (ΙΙ) with murexide.

A very sensitive and selective spectrofluorimetric method has been developed for nickel (ΙΙ) determination in environmental samples. The method is based on measuring the decrease in fluorescence intensity of murexide after nickel (ΙΙ) binding. The intensity of the fluorescence emission peak was measured at ex/em 345/431 nm in several solutions with pH interval 3.0-7.0. The fluorescence intensity decrease was found to be linear in the concentration range of 0.007 mg.L(-1) to 0.1 mg.L(-1) and 0.1 mg.L(-1) to 20 mg.L(-1) of nickel (ΙΙ) by using 10(-4) M murexide at pH 3. The detection limit was found 0.004 mg.L(-1). Relatively large excesses of over 20 cations and anions do not interfere. The method was successfully applied to the analysis of nickel (ΙΙ) in sea, rain and ground water. This method is very precise and accurate (R.S.D. = 0.42% for the determination of 0.05 mg.L-(1) nickel in 10 replicates).

Metal binding to ligands: cadmium complexes with glutathione revisited.

We studied the interaction of gamma-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) with cadmium ions (Cd(2+)) by first performing classical potentiometric pH titration measurements and then turning to additional spectroscopic methods. To estimate the residual concentrations of free cadmium, we studied the competition of glutathione with a Cd(2+)-sensitive dye, either an absorbing dye (murexide) or a fluorescent one (FluoZin-1), and consistent results were obtained with the two dyes. In KCl-containing Tes, Mops, or Tris buffer at pH 7.0 to 7.1 and 37 degrees C (and at a total Cd(2+) concentration of 0.01 mM), results suggest that free cadmium concentration is halved when the concentration of glutathione is approximately 0.05 mM; this mainly reflects the combined apparent dissociation constant for the Cd(glutathione) 1:1 complex under these conditions. To identify the other complexes formed, we used far-UV spectroscopy of the ligand-to-metal charge transfer absorption bands. The Cd(glutathione)(2) 1:2 complex predominated over the 1:1 complex only at high millimolar concentrations of total glutathione and not at low submillimolar concentrations of total glutathione. The apparent conditional constants derived from these spectroscopy results made it possible to discriminate between sets of absolute constants that would otherwise have simulated the pH titration data similarly well in this complicated system. Related experiments showed that although the Cl(-) ions in our media competed (modestly) with glutathione for binding to Cd(2+), the buffers we had chosen did not bind Cd(2+) significantly under our conditions. Our experiments also revealed that Cd(2+) may be adsorbed onto quartz or glass vessel walls, reducing the accuracy of theoretical predictions of the concentrations of species in solution. Lastly, the experiments confirmed the rapid kinetics of formation and dissociation of the UV-absorbing Cd(glutathione)(2) 1:2 complexes. The methods described here may be useful for biochemists needing to determine conditional binding constants for charge transfer metal-ligand complexes under their own conditions.

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.

Calcium binding to metallochromic dyes and calmodulin in the presence of combined, AC-DC magnetic fields.

The possibility that weak, ac and dc magnetic fields in combination may affect binding equilibria of calcium-ions (Ca2+) was investigated with two metallochromic dyes as calcium-binding molecules: murexide and arsenazo III. Calcium-dye equilibria were followed by measuring solution absorbances with a fiber-optic spectrophotometer. A Ca(2+)-arsenazo solution was also used indirectly to monitor the binding of Ca2+ to calmodulin. Parallel, ac and dc magnetic fields were applied to each preparation. The ac magnetic field was held constant during each of a series of experiments at a frequency in the range between 50 and 120 Hz (sine wave) or at 50 pps (square wave) and at an rms flux density in the range between 65 and 156 microT. The dc magnetic field was then varied from 0 to 299 microT at 1.3 microT increments. The magnetic fields did not measurably affect equilibria in the binding of metallochromic dyes or calmodulin to Ca2+.

Intravesicular calcium transient during calcium release from sarcoplasmic reticulum.

The time course of changes in the intravesicular Ca2+ concentration ([Ca2+]i) in terminal cisternal sarcoplasmic reticulum vesicles upon the induction of Ca2+ release was investigated by using tetramethylmurexide (TMX) as an intravesicular Ca2+ probe. Upon the addition of polylysine at the concentration that led to the maximum rate of Ca2+ release, [Ca2+]i decreased monotonically in parallel with Ca2+ release. Upon induction of Ca2+ release by lower concentrations of polylysine, [Ca2+]i first increased above the resting level, followed by a decrease well below it. The release triggers polylysine, and caffeine brought about dissociation of calcium that bound to a nonvesicular membrane segment consisting of the junctional face membrane and calsequestrin bound to it, as monitored with TMX. No Ca2+ dissociation from calsequestrin-free junctional face membranes or from the dissociated calsequestrin was produced by release triggers, but upon reassociation of the dissociated calsequestrin and the junctional face membrane, Ca2+ dissociation by triggers was restored. On the basis of these results, we propose that the release triggers elicit a signal in the junctional face membrane, presumably in the foot protein moiety, which is then transmitted to calsequestrin, leading to the dissociation of the bound calcium; and in SR vesicles, to the transient increase of [Ca2+]i, and subsequently release across the membrane.

Murexide bleaching: a new direct assay method for characterizing reactive oxygen species.

The murexide (5,5'-nitrilodibarbituric acid, monoammonium salt) is an efficient scavenger for superoxide and hydroxyl radicals. When exposed to oxygen radicals, murexide is converted to a colorless alloxan derivative and its absorbance at 520 nm decreases in proportion to the radicals produced. It is used to detect these reactive oxygen species in biochemical systems such as acetaldehyde oxidation by xanthine oxidase and the respiratory burst of polymorphonuclear leukocytes induced by phorbol 12-myristate, 13-acetate. The method was sensitive enough to allow direct monitoring of the production of superoxides from 10(6) phorbol 12-myristate, 13-acetate polymorphonuclear leukocyte-stimulated cells. Moreover, murexide bleaching is inhibited in the presence of radical scavengers, allowing a comparison of their scavenging activities.

Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide.

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the end-pool solutions. The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.27 of indicator was reversibly bound to myoplasmic constituents and the free diffusion constant was 1.75 x 10(-6) cm2/s at 18 degrees C. The shape of the resting absorbance spectrum suggests that a fraction 0.11-0.15 of tetramethylmurexide inside a fiber was complexed with Ca. After action potential stimulation, there was a rapid transient change in indicator absorbance followed by a maintained change of opposite sign. The wavelength dependence of both changes matched a cuvette Ca-difference spectrum. The amplitude of the early peak varied linearly with indicator concentration and corresponded to an average rise in free [Ca] of 17 microM. These rather diverse findings can be explained if the sarcoplasmic reticulum membranes are permeable to Ca-free indicator. Both Ca-free and Ca-complexed indicator inside the sarcoplasmic reticulum would appear to be bound by diffusion analysis and the Ca-complexed form would be detected by the resting absorbance spectrum. The transient change in indicator absorbance would be produced by myoplasmic Ca reacting with indicator molecules that freely diffuse in myoplasmic solution. The maintained signal, which reports Ca dissociating from indicator complexed at rest, would come from changes within the sarcoplasmic reticulum. A method, based on these ideas, is described for separating the two components of the tetramethylmurexide signal. The estimated myoplasmic free [Ca] transient has an average peak value of 26 microM at 18 degrees C. Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143).

Reduction of the metallochromic indicators murexide and tetramethylmurexide to their free radical metabolites by cytoplasmic enzymes and reducing agents.

Murexide underwent reduction by rat liver cytosolic fraction or a hypoxanthine-xanthine oxidase system to produce a free radical metabolite. Reduction of murexide by the freshly prepared cytosolic fraction depended upon the presence of ascorbic acid. N1-Methylnicotinamide, xanthine or hypoxanthine, in that order, could also serve as a source of reducing equivalents for the production of that free radical by the cytosolic fraction. Several thiol compounds (GSH, cysteine, and cysteamine), pyridine nucleotides (NADH, NADPH) and ascorbic acid were also effective in generating the murexide-derived free radical. Tetramethyl murexide was also reduced to its free radical derivative by a hypoxanthine-xanthine oxidase system.