Immobilization of lutetium bisphthalocyanine in nanostructured biomimetic sensors using the LbL technique for phenol detection

This study describes the development of amperometric sensors based on poly(allylamine hydrochloride) (PAH) and lutetium bisphthalocyanine (LuPc(2)) films assembled using the Layer-by-Layer (LbL) technique. The films have been used as modified electrodes for catechol quantification. Electrochemical measurements have been employed to investigate the catalytic properties of the LuPc(2) immobilized in the LbL films. By chronoamperometry, the sensors present excellent sensitivity (20 nA μM(-1)) in a wide linear range (R(2)=0.994) up to 900 μM and limit of detection (s/n=3) of 37.5 × 10(-8)M for catechol. The sensors have good reproducibility and can be used at least for ten times. The work potential is +0.3 V vs. saturated calomel electrode (SCE). In voltammetry measurements, the calibration curve shows a good linearity (R(2)=0.992) in the range of catechol up to 500 μM with a sensitivity of 90 nA μM(-1) and LD of 8 μM.     

Detection of phenolic compounds using impedance spectroscopy measurements

Langmuir-Blodgett (LB) and layer-by-layer films (LbL) of a PPV (p-phenylenevinylene) derivative, an azo compound and tetrasulfonated phthalocyanines were successfully employed as transducers in an “electronic tongue” system for detecting trace levels of phenolic compounds in water. The choice of the materials was based on their distinct electrical natures, which enabled the array to establish a fingerprint of very similar liquids. Impedance spectroscopy measurements were taken in the frequency range from 10 Hz to 1 MHz, with the data analysed with principal component analysis (PCA). The sensing units were obtained from five-layer LB films of (poly[(2-methoxy-5-n-hexyloxy)-p-phenylenevinylene]), OC₁OC₁₈-PPV (poly(2-methoxy,5-(n-octadecyl)-p-phenylenevinylene)), DR (HEMA-co-DR13MA (poly-(hydroxyethylmethacrylate-co-[4′-[[2-(methacryloyloxy)-ethyl]ethylamino]-2-chloro-4-nitroazobenzene]))) and five-bilayer LbL films of tetrasulfonated metallic phthalocyanines deposited onto gold interdigitated electrodes. The sensors were immersed into phenol, 2-chloro-4-methoxyphenol, 2-chlorophenol and 3-chlorophenol (isomers) solutions at 1 × 10⁻⁹ mol L⁻¹, with control experiments carried out in ultra pure water. Samples could be distinguished if the principal component analysis (PCA) plots were made with capacitance values taken at 10³ Hz, which is promising for detection of trace amounts of phenolic pollutants in natural water.     

Development of nanostructured bioanodes containing dendrimers and dehydrogenases enzymes for application in ethanol biofuel cells

This paper describes the use of the electrostatic layer-by-layer (LbL) technique for the preparation of bioanodes with potential application in ethanol/O(2) biofuel cells. More specifically, the LbL technique was employed for immobilization of dehydrogenase enzymes and polyamidoamine (PAMAM) dendrimers onto carbon paper support. Both mono (anchoring only the enzyme alcohol dehydrogenase, ADH) and bi-enzymatic (anchoring both ADH and aldehyde dehydrogenase, AldDH) systems were tested. The amount of ADH deposited onto the Toray? paper was 95 ng cm(-2) per bilayer. Kinetic studies revealed that the LbL technique enables better control of enzyme disposition on the bioanode, as compared with the results obtained with the bioanodes prepared by the passive adsorption technique. The power density values achieved for the mono-enzymatic system as a function of the enzyme load ranged from 0.02 to 0.063 mW cm(-2) for the bioanode containing 36 ADH bilayers. The bioanodes containing a gas diffusion layer (GDL) displayed enhanced performance, but their mechanical stability must be improved. The bi-enzymatic system generated a power density of 0.12 mW cm(-2). In conclusion, the LbL technique is a very attractive approach for enzyme immobilization onto carbon platform, since it enables strict control of enzyme disposition on the bioanode surface with very low enzyme consumption.     

Drastically lowered protein adsorption on microbicidal hydrophobic/hydrophilic polyelectrolyte multilayers

Polyelectrolyte multilayer films assembled from a hydrophobic N-alkylated polyethylenimine and a hydrophilic polyacrylate were discovered to exhibit strong antifouling, as well as antimicrobial, activities. Surfaces coated with these layer-by-layer (LbL) films, which range from 6 to 10 bilayers (up to 45 nm in thickness), adsorbed up to 20 times less protein from blood plasma than the uncoated controls. The dependence of the antifouling activity on the nature of the polycation, as well as on assembly conditions and the number of layers in the LbL films, was investigated. Changing the hydrophobicity of the polycation altered the surface composition and the resistance to protein adsorption of the LbL films. Importantly, this resistance was greater for coated surfaces with the polyanion on top; for these films, the average zeta potential pointed to a near neutral surface charge, thus, presumably minimizing their electrostatic interactions with the protein. The film surface exhibited a large contact angle hysteresis, indicating a heterogeneous topology likely due to the existence of hydrophobic-hydrophilic regions on the surface. Scanning electron micrographs of the film surface revealed the existence of nanoscale domains. We hypothesize that the existence of hydrophobic/hydrophilic nanodomains, as well as surface charge neutrality, contributes to the LbL film's resistance to protein adsorption.     

Layer-by-layer films made from extracellular matrix macromolecules on silicone substrates

The layer-by-layer (LbL) technique has been widely used to produce nanofilms for biomedical applications. Naturally occurring polymers such as ECM macromolecules are attractive candidates for LbL film preparation. In this study, we assessed the build-up of type I collagen (Col1)/chondroitin sulfate (CS) or Col1/Heparin (HN) on polydimethylsiloxane (PDMS) substrates. The build-up was assessed by quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM). Integrin-mediated cell adhesion was assessed by studying the cytoskeletal organization of mammalian primary cells (chondrocytes) seeded on different end layers and number of layers. Data generated from the QCM-D observations showed a consistent build-up of films with more adsorption in the case of Col1/HN. Col1/CS films were stable in media, whereas Col1/HN films were not. AFM analysis showed that the layers were fibrillar in structure for both systems and between 20 and 30 nm thick. The films promoted cell adhesion when compared with tissue culture plastic in serum-free media with cycloheximide. Crosslinking of the films resulted in constrained cell spreading and a ruffled morphology. Finally, beta1 integrin blocking antibodies prevented cell spreading, suggesting that cell adhesion and spreading were mediated mainly by interaction with the collagen fibrils. The ability to construct stable ECM-based films on PDMS has particular relevance in mechanobiology, microfluidics, and other biomedical applications.     

New nanocomposite films for biosensors: layer-by-layer adsorbed films of polyelectrolytes, proteins or DNA

This report describes the construction of ultrathin multicomponent films with an internal structure on the nanometre scale. In the simplest case, the films are built-up by the subsequent adsorption of polyanions and polycations. They can be fabricated on inorganic substrates such as glass, quartz or silicon wafers, or on various organic materials. The polymeric interlayers can incorporate materials with desired electrical or optical properties. The average thickness of the layers can be fine-tuned with Angstrom precision by the addition of suitable salts. They are temperature stable up to at least 200°C and can be laterally structured using conventional photolithographic techniques. The films provide for a well-defined substrate-independent interface for the immobilization of biological macromolecules, such as proteins or DNA, in their active state. The immobilization of streptavidin enables the controlled attachment of any biotinylated molecule with no resulting loss in its biological activity. Area-selective immobilization provides the possibility of built-in quality control for the fabrication of biosensors with separated reference and sample areas on the same substrate.     

Electrostatic adsorption of heme proteins alternated with polyamidoamine dendrimers for layer-by-layer assembly of electroactive films

A novel thin film of heme proteins, including hemoglobin (Hb), myoglobin (Mb), and catalase (Cat), was successfully assembled layer by layer with polyamidoamine (PAMAM) dendrimers on different solid surfaces. At pH 7.0, protonated PAMAM possesses positive surface charges, whereas the proteins have net negative surface charges at pH above their isoelectric points. Thus, layer-by-layer {PAMAM/protein}(n)() films were assembled with alternate adsorption of oppositely charged PAMAM and proteins from their aqueous solutions mainly by electrostatic interaction. The assembly process was monitored by quartz crystal microbalance (QCM), UV-vis spectroscopy, and cyclic voltammetry (CV). The growth of the protein multilayer films was regular and linear, whereas the electroactivity of the films was only extended to a few bilayers. CVs of {PAMAM/protein}(n)() films showed a pair of well-defined and nearly reversible peaks characteristic of the protein heme Fe(III)/Fe(II) redox couples. Although {PAMAM/Hb}(n)() and {PAMAM/Mb}(n)() films showed very similar properties, {PAMAM/Cat}(n)() films displayed different and unique characters. The substrates with biological or environmental significance, such as oxygen, hydrogen peroxide, trichloroacetic acid, and nitrite, were catalytically reduced at {PAMAM/protein}(n)() film electrodes, showing the potential applicability of the films as new types of biosensors or bioreactors based on direct electrochemistry of the proteins. Both the electrochemical and electrocatalytic activity of {PAMAM/protein}(n)() films can be tailored precisely by controlling the number of bilayers or the film thickness.     

Improvement of Transparent Metal Top Electrodes for Organic Solar Cells by Introducing a High Surface Energy Seed Layer

We present highly transparent and conductive silver thin films in a thermally evaporated dielectric/metal/dielectric (DMD) multilayer architecture as top electrode for efficient small molecule organic solar cells. DMD electrodes are frequently used for optoelectronic devices and exhibit excellent optical and electrical properties. Here, we show that ultrathin seed layers such as calcium, aluminum, and gold of only 1 nm thickness strongly influence the morphology of the subsequently deposited silver layer used as electrode. The wetting of silver on the substrate is significantly improved with increasing surface energy of the seed material resulting in enhanced optical and electrical properties. Typically thermally evaporated silver on a dielectric material forms rough and granular layers which are not closed and not conductive below thicknesses of 10 nm. With gold acting as seed layer, the silver electrode forms a continuous, smooth, conductive layer down to a silver thickness of 3 nm. At 7 nm silver thickness such an electrode exhibits a sheet resistance of 19 Ω/□ and a peak transmittance of 83% at 580 nm wavelength, both superior compared to silver electrodes without seed layer and even to indium tin oxide (ITO). Top‐illuminated solar cells using gold/silver double layer electrodes achieve power conversion efficiencies of 4.7%, which is equal to 4.6% observed in bottom‐illuminated reference devices employing conventional ITO. The top electrodes investigated here exhibit promising properties for semitransparent solar cells or devices fabricated on opaque substrates.     

Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10(2) to 10(3) gene copies, which was lowered to 10(0) to 10(1) gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2, 3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2, 3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

The interaction of ascorbate oxidase with L-dopa,L-tyrosine and 3,4-dihydroxycinnamic acid. Evidence for irreversible damage of the enzyme during catechol oxidase activity

The aerobic interaction between ascorbate oxidase and L-tyrosine, L-3,4-dihydroxyphenylalanine or 3,4-dihydroxycinnamic acid in 1:10 molar ratio was followed by optical absorption, CD and EPR spectroscopy in 0.1 M phosphate buffer at pH 5.0. While the spectra of the system ascorbate oxidase—L-tyrosine remain practically unaffected after several hours, indicating that no oxidation of the amino acid occurs in the conditions employed, rather drastic changes can be observed in the spectra of the ascorbate oxidase-catechol systems. In particular, while the optical absorption below 500 nm increases markedly due to the formation of the substrate oxidation products, an irreversible decrease in intensity of the absorption, CD and EPR spectral features associated with the blue copper(II) chromophores indicates that a partial loss of Type 1 copper by ascorbate oxidase has occurred during this secondary catechol oxidase activity. A copper species characterized by weak positive CD activity at 370 nm and EPR signal at intermediate field between those of the Type 2 and Type 1 coppers can be detected in the early stages of the reaction. The irreversible damage undergone by the protein during catechol oxidase activity may have biological significance and accounts for the low yield of purified enzyme obtained when the crude enzyme extract is left in prolonged contact with low molecular weight cell components, rich in σ-diphenolic compounds.     

Colorimetric paper bioassay by horseradish peroxidase for the detection of catechol and resorcinol in aqueous samples

Abstract

Phenolic compounds such as catechol and resorcinol are toxic and persistent pollutants in the aqueous environment. Detection procedures such as chromatographic and spectrophotometric methods are time-consuming and require sophisticated instruments with skilled manpower. Development of a simple, cost effective, portable and disposable paper based biosensor could be a better alternative to the conventional methods. The present study attempted to develop a paper based biosensor by immobilizing horseradish peroxidase enzyme to detect catechol and resorcinol in aqueous samples. Horseradish peroxidase catalyzes the oxidation of phenolic compounds to semiquinones, which on reaction with a chromogen, 3-methyl 2-benzothiazolinone hydrazine (MBTH) gives faint pink to red color depending on the compound and its concentration in the sample is the basis for biosensing application. Different methods of enzyme immobilization on filter paper like physical adsorption, covalent coupling, and polysaccharide entrapment were executed. The performance of the various enzyme immobilization methods was evaluated by analyzing the developed color intensity using ImageJ software. Entrapment technique is the most effective method of immobilizing enzyme on the filter paper that produces the highest color intensity with better stability. The visible limit of detection (LoD) was observed as 0.45?mM (50?mg/L) for catechol and 0.09?mM (10?mg/L) for resorcinol in aqueous samples.     

Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

Morphological and optical characterization of polyelectrolyte multilayers incorporating nanocrystalline cellulose

Aqueous layer-by-layer (LbL) processing was used to create polyelectrolyte multilayer (PEM) nanocomposites containing cellulose nanocrystals and poly(allylamine hydrochloride). Solution-dipping and spin-coating assembly methods gave smooth, stable, thin films. Morphology was studied by atomic force microscopy (AFM) and scanning electron microscopy (SEM), and film growth was characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry, and optical reflectometry. Relatively few deposition cycles were needed to give full surface coverage, with film thicknesses ranging from 10 to 500 nm. Films prepared by spin-coating were substantially thicker than solution-dipped films and displayed radial orientation of the rod-shaped cellulose nanocrystals. The relationship between film color and thickness is discussed according to the principles of thin film interference and indicates that the iridescent properties of the films can be easily tailored in this system.     

Development of Catechol 2,3-Dioxygenase-Specific Primers for Monitoring Bioremediation by Competitive Quantitative PCR

Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primers that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10² to 10³ gene copies, which was lowered to 10⁰ to 10¹ gene copies by hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR with a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.     

Light Absorption and Recycling in Hybrid Metal Halide Perovskite Photovoltaic Devices

The production of highly efficient single‐ and multijunction metal halide perovskite (MHP) solar cells requires careful optimization of the optical and electrical properties of these devices. Here, precise control of CH₃NH₃PbI₃ perovskite layers is demonstrated in solar cell devices through the use of dual source coevaporation. Light absorption and device performance are tracked for incorporated MHP films ranging from ≈67 nm to ≈1.4 µm thickness and transfer‐matrix optical modeling is utilized to quantify optical losses that arise from interference effects. Based on these results, a device with 19.2% steady‐state power conversion efficiency is achieved through incorporation of a perovskite film with near‐optimum predicted thickness (≈709 nm). Significantly, a clear signature of photon reabsorption is observed in perovskite films that have the same thickness (≈709 nm) as in the optimized device. Despite the positive effect of photon recycling associated with photon reabsorption, devices with thicker (>750 nm) MHP layers exhibit poor performance owing to competing nonradiative charge recombination in a “dead‐volume” of MHP. Overall, these findings demonstrate the need for fine control over MHP thickness to achieve the highest efficiency cells, and accurate consideration of photon reabsorption, optical interference, and charge transport properties.     

Imaging and tracking of single GFP molecules in solution

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.     

Reactive thin polymer films as platforms for the immobilization of biomolecules

Spin-coated thin films of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA) on oxidized silicon and gold surfaces were investigated as reactive layers for obtaining platforms for biomolecule immobilization with high molecular loading. The surface reactivity of PNHSMA films in coupling reactions with various primary amines, including amine-terminated poly(ethylene glycol) (PEG-NH2) and fluoresceinamine, was determined by Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and ellipsometry measurements, respectively. The rate constants of PEG-NH2 attachment on the PNHSMA films were found to be significantly increased compared to the coupling on self-assembled monolayers (SAMs) of 11,11'-dithiobis(N-hydroxysuccinimidylundecanoate) (NHS-C10) on gold under the same conditions. More significantly, the PEG loading observed was about 3 times higher for the polymer thin films. These data indicate that the coupling reactions are not limited to the very surface of the polymer films, but proceed into the near-surface regions of the films. PNHSMA films were shown to be stable in contact with aqueous buffer; the swelling analysis, as performed by atomic force microscopy (AFM), indicated a film thickness independent swelling of approximately 2 nm. An increased loading was also observed by surface plasmon resonance for the covalent immobilization of amino-functionalized probe DNA. Hybridization of fluorescently labeled target DNA was successfully detected by fluorescence microscopy and surface plasmon resonance enhanced fluorescence spectroscopy (SPFS), thereby demonstrating that thin films of PNHSMA comprise an attractive and simple platform for the immobilization of biomolecules with high densities.     

Biosensing under an applied voltage using optical waveguide lightmode spectroscopy

An applied dc voltage offers a means of controlling immobilization during biosensor fabrication and detection during biosensing application. We present a method to directly and continuously measure the adsorption of biomacromolecules or other polyelectrolytes, under an applied potential difference, based on optical waveguide lightmode spectroscopy (OWLS). An indium tin oxide (ITO) film of thickness ca. 10 nm coated onto a silicon titanium oxide (STO) waveguiding film serves as the working (sensing) electrode. We observe the effective refractive index of the 0th transverse electric guided mode to increase significantly in the presence of an applied potential due to charging of the interfacial double layer and, possibly, modest electrochemical oxidation. Adsorption from solution onto the ITO electrode is detected by a further increase in the effective refractive index. We achieve accurate detection by employing an optical model in which the STO and ITO layers are combined into a single waveguiding film. No improvement is found using models treating the ITO as a separate layer, either dielectric or conducting. Using this method, we find the adsorption of human serum albumin and horse heart cytochrome c to be considerably enhanced in the presence of an applied potential exceeding 1 V. We attribute this behavior to adsorption at positions on the protein molecules of complementary charge.     

The catalytic effect of tyrosinase upon oxidation of 2-hydroxyestradiol in presence of catechol

The hydroxylating activity of mushroom tyrosinase has been utilized for over a decade in the preparation of 2-hydroxyestradiol from estradiol, yet this same enzyme is known to function as an oxidant of o-dihydric compounds to the corresponding o-quinones. It was questioned why catechol estrogens do not react further, particularly since the tyrosinase activity (hydroxylating) is exceeded many fold by the diphenol oxidase activity of the enzyme. This report describes that the catechol estrogen will react in presence of enzyme but only if catechol is also present. Diphenol oxidase activity was measured either by the polarographic oxygen-utilization technique or by changes in the absorption spectrum at 206 and 256 nm. The enzyme activity was standardized with catechol (Km = 5.2 X 10(-4) M). The steroid did not react with the enzyme if catechol was absent. With catechol, the steroid reacted rapidly and completely (Km = 4.2 X 10(-4) M). The consumption of oxygen with catechol and 2-hydroxyestradiol was additive and stoichiometric, 1 g-atom oxygen/mol of either substrate. Kinetic analysis shows that catechol functions as an activator of the tyrosinase.     

Studies on rheomelanins. V. Hemolysis associated with the transformation of catechol into rheomelanin in human blood.

Incubation of 2 mg amounts of catechol in 5 ml samples of heparinated blood plasma from four subjects at 38 degrees C for 24 h produced plasma-soluble rheomelanins. These solutions had the brown color and the yellow-green fluorescence in ultraviolet light of 366 nm of other rheomelanins. Their differential ultraviolet and visible spectra showed a rheomelanin absorption maximum at 344 nm. Paper chromatograms of the rheomelanin-plasma solutions in 5% methanol-95% water showed elongated spots of rheomelanins with RF values of 0.82, on Whatman No. 1 paper. Using heparinated distilled water adjusted to pH 7.4 with sodium bicarbonate instead of human blood plasma gave markedly different findings from those obtained with the plasma rheomelanin solutions. Incubation of 4 mg amounts of catechol in 10 ml samples of heparinated whole blood from four subjects for 24, 32 and 48 at 38 degrees C produced rheomelanins as found in the plasma separated from the blood after incubation. The differential ultraviolet and visible spectra of these solutions revealed hemolysis caused by the catechol rheomelanins; this was more marked with longer incubations. The hemolysis was manifested by two absorption peaks at about 270 and 400 nm. Paper chromatography revealed the brown elongated spots of catechol rheomelanins with an RF value of 0.82. Other spots owing to the products of hemolysis were also present.