A method for removal of trace iron contamination from biological buffers

Laboratory chemicals and reagents normally contain trace amounts of iron salts sufficient to catalyse free radical reactions. This iron contamination can be removed from buffers and reagents using a dialysis sac containing a high-affinity iron-binding protein like conalbumin or transferrin without altering the pH value of the fluid.     

Effect of metal ion concentration on a biological reactor

Saccharomyces cerevisiae (yeast) cells were employed as a source of alcohol dehydrogenase in the NAD(+)-to-NADH reaction. The cells were immobilized in calcium alginate monofilament fibers and used in a biological reactor. The alginate could not be heat sterilized since temperatures above 80 degrees C caused the polymer chains to degrade. The same proved true for the high pH necessary for the reaction, but the alginate strength was increased by Ba(2+) solution treatment. X-ray probe analysis showed that about 30% of the Ca(2+) sites exchanged with the Ba(2+) ions. The Ba(2+) ions (as well as the Ca(2+) ions) permeabilized the cells and increased the reaction rate. Long term trials showed that Ba(2+) ions were slowly elutriated from the fiber biocatalyst, causing a drop in reaction rate. The trend certainly was reversible as far as the fiber was concerned. It is assumed that the permeabilization of the cells by the Ba(2+) ions was a reversible process.     

Interference of Good's buffers and other biological buffers with protein determination

Interference of low concentrations of Hepes and other buffers commonly used in protein determination was studied. The data show that some of these buffers interfere to differing degrees with protein determination according to the Lowry method. A study of the structure-interference relationship suggests that the group ethanolamine is involved in this interference. No interference was observed when protein was measured using bicinchonic acid at the same concentration as the Lowry reagent.     

Bound and determined: a computer program for making buffers of defined ion concentrations.

A computer program that allows the preparation of buffers containing known concentrations of metal-ligand complexes at defined pH values and temperatures is described. Ligands are defined as compounds that bind metals and may include AMP, ADP, ATP, GMP, GDP, GTP, EGTA, EDTA, BAPTA, phosphate, sulfate, chloride, monocarboxylic acids, dicarboxylic acids, organophosphates, and/or citric acid. Metals may include sodium, potassium, magnesium, calcium, and/or manganese. The program uses association constants corrected for temperature and ionic strength so that solutions between 0 and 40 degrees C and between pH values of 4 and 10 can be defined. The program can perform the following: (i) calculate the concentration of all metal-ligand complexes when total metal and total ligand concentrations are known, (ii) calculate the concentration of metal ion required to make a solution of known free metal ion concentration when total ligand concentrations are known, (iii) calculate the concentration of ligand required to make a solution of known free metal ion concentration when total metal concentrations are known, and (iv) calculate the total concentrations of metal and ligand required to make a buffer of known metal-ligand concentration. Options i-iii are useful for making buffers of defined free metal ion concentrations; option iv is useful for making buffers of defined metal-nucleotide concentrations.     

Design and applications of methods for fluorescence detection of iron in biological systems

Fluorescence metalosensors provide a means to detect iron in biological systems that is versatile, economical, sensitive and of a high-throughput nature. They rely on relatively high-affinity iron-binding carriers conjugated to highly fluorescent probes that undergo quenching after metal complexation. Metal specificity is determined by probes containing either an iron-binding moiety of high affinity (type A) or of relatively lower affinity (type B) used in combination with a strong specific iron chelator. Due to the heterogeneous nature of biological systems, the apparent metal-binding affinity and complexation stoichiometry ought to be specifically defined. Fluoresceinated moieties coupled to metal-binding cores detect Fe at sub-micromolar concentrations and even sub-microlitre volumes (i.e. cells). Although an ideal probe should also be specific for a particular oxidation state of iron, in physiological conditions that property might be difficult to attain. Quantification of labile iron in cells has relied on the ability of permeant iron chelators to restore the fluorescence of probes quenched by intracellular Fe. Modern design of probes aims to (a) improve probe targeting to specific cell compartments and (b) create probes that respond to metal binding by signal enhancement.     

An ion exchange method to determine free metal concentrations, adapted for use in biological fluids: methods and determination of (Mg2+)]

An ion exchange method for measuring concentrations of free (ionized) metal ions and its application to the determination of [Mg2+] is described. A surface sulfonated polystyrene material is used as the "twodimensional" cation exchanger. The sample-volume is 1 ml. About 50 determinations can be performed within 1 hr having a standard error of +/- (2-4)% in the optimal range of measurement. Advantages and disadvantages of the method compared with other ones are demonstrated and discussed. Free Mg2+ ion concentrations were measured in solutions containing pyrophosphate as well as haemoglobin and compared with those which were determined by equilibrium calculation or ultrafiltration.     

The effect of various dietary zinc concentrations on the biological interactions of zinc,copper, and iron in rats

Three groups (14 rats each) were fed one of the following diets for 8 wks: a control purified basal diet containing 12 ppm zinc, 5 ppm copper, and 35 ppm iron; the basal diet with less than 2 ppm zinc; or the basal diet supplemented with 1000 ppm zinc. Rats fed the zinc-deficient diet had decreased weight gain, moderate polydipsia, and intermittent mild diarrhea. The zinc-supplemented rats had a cyclical pattern of food intake and weight loss from weeks 5 to 8. Tissue concentrations suggest that zinc and copper were not mutually antagonistic with chronic dietary imbalances. If tissue element concentrations reflected intestinal uptake, then competition and/or inhibition of intestinal uptake occurred between zinc and iron. The fluctuations in tissue element concentrations that occurred with increased duration of the study were at variance with previous studies of shorter time periods. The dietary proportions of zinc, copper, and iron appear to influence zinc, copper, and iron metabolism at the intestinal and cellular transport levels over a given period of time.     

Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is essential for substrate binding and catalysis

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.     

A review of fluorescence methods for assessing labile iron in cells and biological fluids

A variety of biochemical, pharmacological, and toxicological properties have been attributed to labile forms of iron that are associated with cells or with biological fluids. Unlike the major fraction of bioiron which is protein bound, the labile bioiron is chelatable and therefore amenable for detection by metal-sensing devices that are coupled to chelation moieties. The present review deals with the detection of various labile forms of iron present in living cells and in fluids of biological interest, in health and disease. The main focus of the review is on the design and application of fluorescent probes as analytical tools for assessing labile iron and iron transport mechanisms and the efficiency of iron chelators in solution and in cellular systems.     

Significance of metal ions in galactose-1-phosphate uridylyltransferase: an essential structural zinc and a nonessential structural iron.

Galactose-1-phosphate uridylyltransferase (GalT) catalyzes the reversible transformation of UDP-glucose and galactose-1-phosphate (Gal-1-P) into UDP-galactose and glucose-1-phosphate (Glc-1-P) by a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP-enzyme). GalT is a metalloenzyme containing 1.2 mol of zinc and 0.7 mol of iron/mol of subunits [Ruzicka, F. J., Wedekind, J. E., Kim, J., Rayment, I., and Frey, P. A. (1995) Biochemistry 34, 5610-5617]. The zinc site lies 8 A from His 166 in active site, and the iron site lies 30 A from the active site [Wedekind,J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. Zinc is coordinated in tetrahedral geometry by Cys 52, Cys 55, His 115, and His 164. His 164 is part of the highly conserved active-site triad His 164-Pro 165-His 166, in which His 166 is the nucleophilic catalyst. Iron is coordinated in square pyramidal geometry with His 296, His 298, and Glu 182 in bidentate coordination providing the base ligands and His 281 providing the axial ligand. In the present study, site-directed mutagenesis, kinetic, and metal analysis studies show that C52S-, C55S-, and H164N-GalT are 3000-, 600-, and 10000-fold less active than wild-type. None of the variants formed the UMP-enzyme in detectable amounts upon reaction with UDP-Glc in the absence of Gal-1-P. Their zinc content was very low, and the zinc + iron content was about 50% of that for wild-type GalT. Mutation of His 115 to Asn 115 resulted in decreased activity to 2.9% of wild-type, with retention of zinc and iron. In contrast to the zinc-binding site, Glu 182 in the iron site is not important for enzymatic activity. The variant E182A-GalT displayed about half the activity of wild-type GalT, and all of the active sites underwent uridylylation to the UMP-enzyme, similar to wild-type GalT, upon reaction with UDP-Glc. Metal analysis showed that while E182A-GalT contained 0.9 equiv of zinc/subunit, it contained no iron. The residual zinc can be removed by dialysis with 1,10-phenanthroline, with the loss in activity being proportional to the amount of residual zinc. It is concluded that the presence of zinc is essential for maintaining GalT function, whereas the presence of iron is not essential.     

Modification of radiation response by metal complexes: a review with emphasis of nonplatinum studies

There is a need to develop compounds which alter the effects of radiation, particularly in the hypoxic radioresistant cell, following the limited success to date of the electron-affinic nitroimidazoles. The chemistry of transition metals is briefly outlined to point out certain aspects which might be exploited in the design of radiosensitizers. The best known clinical example of a metal complex which enhances the effect of radiation in hypoxic cells is the successful antineoplastic cisplatin. Past studies on enhancement of radiation damage by complexes of metals other than platinum, mainly in bacterial spores and bacterial and mammalian cells, have been summarized according to the metal used. The many mechanisms by which metal complexes could interact with radiation are outlined, and examples are given where possible. This survey emphasizes the need for a systematic study of the effect of metal/ligand variation on radiosensitization with regard to mechanisms of action to assess the potential of these compounds as radiosensitizers. Metal complexes offer many advantages, both for the study of mechanisms by which radiation kills cells and for drug development.     

Electromagnetic hypersensitivity: biological effects of dirty electricity with emphasis on diabetes and multiple sclerosis

Dirty electricity is a ubiquitous pollutant. It flows along wires and radiates from them and involves both extremely low frequency electromagnetic fields and radio frequency radiation. Until recently, dirty electricity has been largely ignored by the scientific community. Recent inventions of metering and filter equipment provide scientists with the tools to measure and reduce dirty electricity on electrical wires. Several case studies and anecdotal reports are presented. Graham/Stetzer (GS) filters have been installed in schools with sick building syndrome and both staff and students reported improved health and more energy. The number of students needing inhalers for asthma was reduced in one school and student behavior associated with ADD/ADHD improved in another school. Blood sugar levels for some diabetics respond to the amount of dirty electricity in their environment. Type 1 diabetics require less insulin and Type 2 diabetics have lower blood sugar levels in an electromagnetically clean environment. Individuals diagnosed with multiple sclerosis have better balance and fewer tremors. Those requiring a cane walked unassisted within a few days to weeks after GS filters were installed in their home. Several disorders, including asthma, ADD/ADHD, diabetes, multiple sclerosis, chronic fatigue, fibromyalgia, are increasing at an alarming rate, as is electromagnetic pollution in the form of dirty electricity, ground current, and radio frequency radiation from wireless devices. The connection between electromagnetic pollution and these disorders needs to be investigated and the percentage of people sensitive to this form of energy needs to be determined.     

Molecular and biochemical aspects of rhizobacterial ecology with emphasis on biological control

The rhizosphere is the narrow zone of soil surrounding the root that is subject to influence by the root. Rhizobacteria are plant-associated bacteria that are able to colonize and persist on roots. An understanding of the ecology of a microorganism is a fundamental requirement for the introduction of a microbial inoculant into the open environment. This is particularly true for biological control of root pathogens in the rhizosphere, where one is actively seeking to alter the ecological balance so as to favour growth of the host plant and to curtail the development of pathogens. Some strains of plant growth-promoting rhizobacteria can effectively colonize plant roots and protect plants from diseases caused by a variety of root pathogens and growth promotion of plants through direct stimulation of growth hormone. Such beneficial or plant health-promoting strains are emerging as promising biocontrol agents. They are suitable as soil inoculants either individually or in combination and may be compatible with current chemical pesticides. Considerable progress has been achieved using molecular genetic techniques to elucidate the important microbial factors or genetic traits involved in the suppression of fungal root diseases. Strategies utilizing molecular genetic techniques have been developed to complement the ongoing research ranging from the characterization and genetic improvement of a selected biocontrol agent to the measurement of its persistence and dispersal. Finally, biocontrol is considered as part of a disease control strategy like integrated pest management which offers a successful approach for the deployment of both agro-chemicals and biocontrol agents.     

Divalent cations as modulators of neuronal excitability: emphasis on copper and zinc

Based on indirect evidence, a role for synaptically released copper and zinc as modulators of neuronal activity has been proposed. To test this proposal directly, we studied the effect of copper, zinc, and other divalent cations on voltage-dependent currents in dissociated toad olfactory neurons and on their firing rate induced by small depolarizing currents. Divalent cations in the nanomolar range sped up the activation kinetics and increased the amplitude of the inward sodium current. In the micromolar range, they caused a dose dependent inhibition of the inward Na+ and Ca2+ currents (INa and ICa) and reduced de amplitude of the Ca2+-dependent K+ outward current (ICa-K). On the other hand, the firing rate of olfactory neurons increased when exposed to nanomolar concentration of divalent cations and decreased when exposed to micromolar concentrations. This biphasic effect of divalent cations on neuronal excitability may be explained by the interaction of these ions with high and low affinity sites in voltage-gated channels. Our results support the idea that these ions are normal modulators of neuronal excitability.