Melanin standard method: particle description.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.     

Melanin Standard Method: Titrimetric Analysis

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48–53, 1992).     

Melanin Standard Method: Empirical Formula

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.     

Melanin Standard Method: Empirical Formula 2

Natural melanins are composed of two distinct portions; a protein fraction and a chromophoric backbone. There is no unequivocal evidence for covalent bonding between these two fractions, and standard protocols used in protein purification have failed to separate the protein fraction from the chromophoric fraction. In order to study the chromophoric backbone, many workers have resorted to harsh isolation and purification protocols that are now known to degrade and damage the chromophoric portion. These artifactual melanin preparations are poor models for valid chemical, physical, and biological studies. We have developed a mild isolation and purification protocol for melanins that takes into consideration both the particulate nature of natural melanins and the stability characteristics of the chromophoric fraction. Mathematical factoring of the quantitative amino acid data into the elemental analysis was used to obtain the empirical formula of the chromophoric backbone of melanins. The analyses have shown that melanins from various sources have significantly different amino acid compositions and contents, molar C/N ratios, and empirical formulae. This method successfully differentiates melanins from a variety of sources, namely, human hair, Sepia officinalis, Sigma Chemical Company (cat. no. M8631), autoxidation of dopa, and from the feathers of Rhode Island Red chickens. Analytical results from these studies are presented and discussed.     

The Photochemical and Surface-Active Properties of Melanins Isolated from Some Black Fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < < 400 nm) and in the blue–violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm². The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Effects on Mitochondrial K Flux of pH,K Concentration,and N-Ethyl Maleimide

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K⁺ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH^? influx or H⁺ efflux. The dependence of the unidirectional K⁺ influx on the external K⁺ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V_max of the K⁺ influx without significantly altering the apparent K_m for K⁺. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial P_i/OH^? exchange mechanism. N-Ethyl maleimide decreases the apparent V_max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external P_i concentration may stimulate the K⁺ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous P_i and external OH^?. An apparent first-order dependence of the K⁺ influx on the external OH^? concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH^? as a cosubstrate of the K⁺ transport mechanism.     

Isolation,purification and physicochemical characterization of water‐soluble Bacillus thuringiensis melanin

Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water‐insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water‐soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water‐soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water‐soluble melanin from this organism has an isoelectric point (pI = 3.0–3.2) and was purified optimally by adsorbtion using the IA‐1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra‐red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS‐PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g = 2.006. The concentration of paramagnetic centers in melanin is 0.21 × 10¹⁸ spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.     

Transport of alkali cations through thin lipid membranes by (222)C10-Cryptand,an ionizable mobile carrier

Summary The kinetics of K⁺ and Na⁺ transport across the membrane of large unilamellar vesicles (L.U.V.) were compared at two pH's, with two carriers: (222)C ₁₀-cryptand (diaza-1, 10-decyl-5-hexaoxa-4,7,13,16,21,24-bicyclo[8.8.8.]hexacosane) and valinomcyin, i.e. an ionizable macrobicyclic amino polyether and a neutral macrocyclic antibiotic. The rate of cation transport by (222)C₁₀ saturated as cation and carrier concentrations rose. The apparent affinity of (222)C₁₀ for K⁺ was higher and less pH dependent than that for Na⁺ but resembled the affinity of valinomycin for K⁺. The efficiency of (222)C₁₀ transport of K⁺ decreased as the pH fell and the carrier concentration rose, and was about ten times lower than that of valinomycin. Noncompetitive K⁺/Na⁺ transport selectivity of (222)C₁₀ decreased as pH, and cation and carrier concentrations rose, and was lower than that of valinomycin. Transport of alkali cations by (222)C₁₀ and valinomycin was noncooperative. Reaction orders in cationn(S) and carrierm(M) varied with the type of cation and carrier and were almost independent of pH;n(S) andm(M) were not respectively dependent on carrier or cation concentrations. The apparent estimated constants for cation translocation by (222)C₁₀ were higher in the presence of Na⁺ than of K⁺ due to higher carrier saturation by K⁺, and decreased as pH and carrier concentration increased. Equilibrium potential was independent of the nature of carrier and transported cation. Results are discussed in terms of the structural, physicochemical and electrical characteristics of carriers and complexes.     

Melanin standard method: empirical formula.

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.     

Induction of Na+ / K+ exchange in swollen heart mitochondria

Heart mitochondria swollen passively in nitrate salts contract in a respiration-dependent reaction which can be attributed to an endogenous cation/H⁺ exchange component (or components). The rate of contraction increases with increased extent of passive swelling in both Na⁺ and K⁺ salts. Since nearly constant internal cation concentrations are maintained during osmotic swelling, this result suggests that both Na⁺/H⁺ and K⁺/H⁺ exchange is enhanced by increased matrix volume. Endogenous Mg²⁺ is also lost with increased matrix volume, and this observation, in conjunction with other evidence available in the literature, suggests that monovalent cation/H⁺ exchanges may be regulated by divalent cations. Passive exchange of Na⁺/K⁺,⁴²K⁺/K⁺, and²⁴Na⁺/Na⁺ can be readily demonstrated in mitochondria swollen in nitrate. All these exchanges are low or not detectable in unswollen control mitochondria, and it appears that they are manifestations of the activated cation/H⁺ component (or components) functioning in the absence of pH.     

Thallium interaction with the gastric (K,H)-ATPase

Summary The gastric (K,H)-ATPase has been shown to catalyze an electroneutral H⁺ for K⁺ exchange. Tl⁺ is able to substitute for K⁺ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 m as compared to about 870 m for K⁺. The ability of Tl⁺ to participate in transport is shown by the development of pH gradients in the presence of Tl⁺ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl⁺ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl⁺ concentrations above 3.0mm. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl⁺ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl⁺ concentrations, following the initial Tl⁺ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl⁺ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t _1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl⁺ concentrations in excess of 1 to 4mm are able to dissipate intravesicular proton gradients by an electrically coupled H⁺ for Tl⁺ exchange. Thus, although Tl⁺ is able to activate the gastric ATPase by mimicking K⁺, the permeability of this cation in lipid bilayers tends to uncouple H⁺ transport at concentrations high enough to generate detectable proton gradients.     

Metal–ion interactions and the structural organization of Sepia eumelanin

The structural organization of melanin granules isolated from ink sacs of Sepia officinalis was examined as a function of metal ion content by scanning electron microscopy and atomic force microscopy. Exposing Sepia melanin granules to ethelenediaminetetraacetic acid (EDTA) solution or to metal salt solutions changed the metal content in the melanin, but did not alter granular morphology. Thus ionic forces between the organic components and metal ions in melanin are not required to sustain the natural morphology once the granule is assembled. However, when aqueous suspensions of Sepia melanin granules of varying metal content are ultra‐sonicated, EDTA‐washed and Fe‐saturated melanin samples lose material to the solution more readily than the corresponding Ca(II) and Mg(II)‐loaded samples. The solubilized components are found to be 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐rich constituents. Associated with different metal ions, Na(I), Ca(II) and Mg(II) or Fe(III), these DHICA‐rich entities form distinct two‐dimensional aggregation structures when dried on the flat surface of mica. The data suggest multiply‐charged ions play an important role in assisting or templating the assembly of the metal‐free organic components to form the three‐dimensional substructure distributed along the protein scaffold within the granule.     

The use of phospholipid-impregnated millipore filters for recording nonelectrogenic cation flows in the presence of Me/nH exchangers

It has been shown that with a cation (K⁺, Na⁺, Ca²⁺) concentration gradient on a Millipore filter impregnated with a decane solution of phospholipid, in the presence of a Meⁿ⁺/nH⁺ exchanger (nigericin, monensin, A23187), addition of a protonophore induces the formation of an electric potential positively charged on the side where the concentration of the cation is lower. The formation of the potential is induced by the hydrogen ion concentration gradient in the filter and in the unstirred layers as a result of the Meⁿ⁺/nH⁺ exchange. In such a system, with a pH gradient on the filter in the presence of monensin and valinomycin, a potential is generated with the plus on the side of the lower concentration of hydrogen. The effect is the result of the formation of a potassium ion concentration gradient in the unstirred layers in the course of the K⁺/H⁺ exchange. It is concluded that phospholipid-impregnated filters can be used for search and identification of electroneutral membrane ionophores of the Meⁿ⁺/nH⁺ exchanger type.     

Scorpion neurotoxin - a presynaptic toxin which affects both Na+ and K+ channels in axons.

A neurotoxin from the venom of the scorpion, $\text{Androctonus australis Hector}$, affects the closing of the Na⁺ channel and the opening of the K⁺ channel in giant axons of crayfish and lobster nerves. It blocks both Na⁺ and K⁺ conductances in $\text{Sepia}$ giant axons. Dose-response curves are markedly cooperative with all types of axons. Apparent dissociation constants for the receptor-toxin complexes are 0.25 μM, 0.7 μM and 2–4 μM for the crayfish, lobster and $\text{Sepia}$ axons, respectively. This toxin will be probably a useful tool for biochemical investigation of Na⁺ and K⁺ channels.     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Induction of passive monovalent cation-exchange activity in heart mitochondria by depletion of endogenous divalent cations

Depletion of mitochondrial divalent cations by addition of the ionophore A23187 results in a marked increase in passive ${}^{42}\text{K}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange activity. The exchange is activated by increasing pH and temperature and inhibited by added divalent cations. The reaction is independent of the amount of A23187 present, but depends on the concentration of external K⁺ (K_m = 25 mm). Intramitochondrial ⁴²K⁺ in cation-depleted mitochondria exchanges passively with external Na⁺ and Li⁺, but not with choline⁺. The evidence suggests that removal of mitochondrial divalent cations by A23187 activates the endogenous $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange component of the mitochondrion and that the activated exchanger promotes cation/cation exchange in the absence of a metabolic pH gradient.     

Phthaloyl‐coenzyme A decarboxylase from Thauera chlorobenzoica: the prenylated flavin‐, K+‐ and Fe2+‐dependent key enzyme of anaerobic phthalate degradation

The degradation of the industrially produced and environmentally relevant phthalate esters by microorganisms is initiated by the hydrolysis to alcohols and phthalate (1,2‐dicarboxybenzene). In the absence of oxygen the further degradation of phthalate proceeds via activation to phthaloyl‐CoA followed by decarboxylation to benzoyl‐CoA. Here, we report on the first purification and characterization of a phthaloyl‐CoA decarboxylase (PCD) from the denitrifying Thauera chlorobenzoica. Hexameric PCD belongs to the UbiD‐family of (de)carboxylases and contains prenylated FMN (prFMN), K⁺ and, unlike other UbiD‐like enzymes, Fe²⁺ as cofactors. The latter is suggested to be involved in oxygen‐independent electron‐transfer during oxidative prFMN maturation. Either oxidation to the Fe³⁺‐state in air or removal of K⁺ by desalting resulted in >92% loss of both, prFMN and decarboxylation activity suggesting the presence of an active site prFMN/Fe²⁺/K⁺‐complex in PCD. The PCD‐catalysed reaction was essentially irreversible: neither carboxylation of benzoyl‐CoA in the presence of 2 M bicarbonate, nor an isotope exchange of phthaloyl‐CoA with ¹³C‐bicarbonate was observed. PCD differs in many aspects from prFMN‐containing UbiD‐like decarboxylases and serves as a biochemically accessible model for the large number of UbiD‐like (de)carboxylases that play key roles in the anaerobic degradation of environmentally relevant aromatic pollutants.     

Role of calcium in melanosome aggregation withinLabeo melanophores

In isolated scale melanophores ofLabeo rohita the melanosome aggregating effect of K⁺ was inhibited in Ca²⁺ deprived medium. Moreover, the Ca²⁺-antagonists, verapamil and lanthanum inhibited the action of K⁺ in concentration dependent manner. The elevation of extracellular Ca²⁺ could compromise the verapamil induced inhibition in a concentration dependent manner. The cation Ca²⁺ appeared to be required only for K+ -induced aggregation and not melanosome aggregationper se, as in this fish adrenaline and melanin concentrating hormone effectively caused aggregation of melanosomes in Ca²⁺ free medium     

Study of photochemical and surface-active melanins of some representatives of black fungi

The absorption spectra of melanins isolated from some black ascomycetes, as well as of synthetic melanin and natural melanin from Sepia officinalis, were recorded in the long-wavelength ultraviolet region A (320 nm < lambda < 400 nm) and in the blue-violet region of the electromagnetic spectrum at illumination intensities varying from 0.02 to 1 mW/cm2. The photochemical properties of fungal melanins were found to be dependent on both the producing strain and the conditions of its cultivation. The fungal melanins are more susceptible to photomodification and more biologically active than the synthetic melanin, indicating that these properties may be related. The data obtained suggest that the fungal melanins susceptible to photomodification possess higher biological activity than commercial melanins.     

Calcium-dependent K current in plasma membranes of dermal cells of developing bean cotyledons

In developing seeds of bean (Phaseolus vulgaris L.), phloem‐imported assimilates (largely sucrose and potassium) are released from coats to seed apoplasm and subsequently retrieved by the dermal cell complexes of cotyledons. To investigate the mechanisms of K⁺ uptake by the cotyledons, protoplasts of dermal cell complexes were isolated and whole‐cell currents across their plasma membranes were measured with the patch‐clamp technique. A weakly rectified cation current displaying a voltage‐dependent blockade by external Ca²⁺ and acidic pH, dominated the conductance of the protoplasts. The P haseolus v ulgaris Cotyledon Dermal‐cell pH and Calcium‐dependent Cation Conductance (Pv‐CD‐pHCaCC) was highly selective for K⁺ over Ca²⁺ and Cl^–. For K⁺ current through Pv‐CD‐pHCaCC a sigmoid shaped current–voltage (I–V) curve was observed with negative conductance at voltages between ?200 and ?140 mV. This negative K⁺ conductance was Ca²⁺ dependent. With other univalent cations (Na⁺, Rb⁺, NH₄⁺) the currents were smaller and were not Ca²⁺ dependent. Reversal potentials remained constant when external K⁺ was substituted with these cations, suggesting that Pv‐CD‐pHCaCC channels were non‐selective. The Pv‐CD‐pHCaCC would provide a pathway for K⁺ and other univalent cation influx into developing cotyledons. These cation influxes could be co‐ordinated with sucrose influx via pH and Ca²⁺dependence.