Effect of Humid Air Exposed to IR Radiation on Enzyme Activity.

Water vapor absorbs well in the infra-red region of the electromagnetic spectrum. Absorption of radiant energy by water or water droplets leads to formation of exclusion zone water that possesses peculiar physico-chemical properties. In the course of this study, normally functioning and damaged alkaline phosphatase, horseradish peroxidase and catalase were treated with humid air irradiated with infrared light with a wavelength in the range of 1270 nm and referred to as coherent humidity (CoHu). One-minute long treatment with CoHu helped to partially protect enzymes from heat inactivation, mixed function oxidation, and loss of activity due to partial unfolding. Authors suggest that a possible mechanism underlying the observed effects involves altering the physicochemical properties of aqueous media while treatment of the objects with CoHu where CoHu acts as an intermediary.

An anthraquinone-enzyme-peptide hybrid as a photo-switchable enzyme.

A purpose-designed anthraquinone-horseradish peroxidase (HRP)-cell penetrating peptide hybrid 1 showed high and effective cell permeability. Furthermore, 1 exhibited enzymatic activity without photo-irradiation, whereas significant self-degradation and loss of enzymatic activity occurred upon photo-irradiation in living cells.

Effect of pulsed light on activity and structural changes of horseradish peroxidase.

The objective of this research was to investigate the effects of pulsed light on the activity and structure of horseradish peroxidase in buffer solution. Enzyme residual activities were measured. Surface topography, secondary, and tertiary structures of horseradish peroxidase were determined using atomic force microscopy (AFM), Raman spectroscopy, and fluorescence spectroscopy, respectively. Results showed that a complete inactivation of horseradish peroxidase was achieved by application of 10 pulses of pulsed light treatment at an intensity of 500J/pulse. The AFM analysis revealed that the aggregation of enzyme protein increased and surface roughness decreased with the increase in the treatment time. Fluorescence and Raman spectroscopy analysis exhibited that pulsed light destroyed the tertiary and secondary protein structures. The ß-sheet composition was decreased while ß-turn and random coils were increased. Pulsed light could effectively inactivate horseradish peroxidase by destroying the secondary and tertiary structures of protein in the active center of the enzyme.

Magnetosomes extracted from Magnetospirillum magneticum strain AMB-1 showed enhanced peroxidase-like activity under visible-light irradiation.

Magnetosomes are intracellular structures produced by magnetotactic bacteria and are magnetic nanoparticles surrounded by a lipid bilayer membrane. Magnetosomes reportedly possess intrinsic enzyme mimetic activity similar to that found in horseradish peroxidase (HRP) and can scavenge reactive oxygen species depending on peroxidase activity. Our previous study has demonstrated the phototaxis characteristics of Magnetospirillum magneticum strain AMB-1 cells, but the mechanism is not well understood. Therefore, we studied the relationship between visible-light irradiation and peroxidase-like activity of magnetosomes extracted from M. magneticum strain AMB-1. We then compared this characteristic with that of HRP, iron ions, and naked magnetosomes using 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate in the presence of H2O2. Results showed that HRP and iron ions had different activities from those of magnetosomes and naked magnetosomes when exposed to visible-light irradiation. Magnetosomes and naked magnetosomes had enhanced peroxidase-like activities under visible-light irradiation, but magnetosomes showed less affinity toward substrates than naked magnetosomes under visible-light irradiation. These results suggested that the peroxidase-like activity of magnetosomes may follow an ordered ternary mechanism rather than a ping-pong mechanism. This finding may provide new insight into the function of magnetosomes in the phototaxis in magnetotactic bacteria.

Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination.

Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H(2)O(2) to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption. Longer UV illumination time leads to lower T(m) values as well as helical content loss. Prolonged UV illumination and heme irradiation at 403 nm has a pronounced effect on the fluorescence quantum yield correlated with changes in the prosthetic group pocket, leading to a pronounced decrease in the heme's Soret absorbance band. Analysis of the picosecond-resolved fluorescence emission of horseradish peroxidase A2 with streak camera shows that UV illumination induces an exponential change in the preexponential factors distribution associated to the protein's fluorescence lifetimes, leading to an exponential increase of the mean fluorescence lifetime. Illumination of aromatic residues and of the heme group leads to changes indicative of heme leaving the molecule and/or that photoinduced chemical changes occur in the heme moiety. Our studies bring new insight into light-induced reactions in proteins. We show how streak camera technology can be of outstanding value to follow such ultrafast processes and how streak camera data can be correlated with protein structural changes.

Quenchers induce wavelength dependence on protein fluorescence lifetimes.

We have analysed the picosecond resolved fluorescence emission decay of horseradish peroxidase A2 and of HEW lysozyme acquired with a streak camera. Analyses of the fluorescence decay data of both proteins revealed that the dynamics of the decay is dependent on the emission wavelength. Our data strongly indicates that resonance energy transfer occurring between aromatic residues and different protein fluorescence quencher groups, and the nature of the quencher groups, are the causes of the observed wavelength dependent mean lifetime distribution. Using the global analysis data to calculate the fluorescence mean lifetime at each wavelength revealed that for lysozyme, the mean fluorescence lifetime increased with observation wavelength, whereas the opposite was the case for peroxidase. Both proteins contain strong fluorescence quencher groups located in close spatial proximity to the protein's aromatic residues. Lysozyme contains disulfide bridges as the main fluorescence quencher whereas peroxidase contains a heme group. Both for lysozyme and horseradish peroxidase there is a clear correlation between the observed fluorescence mean lifetime of the protein at a particular emission wavelength and the respective quencher's extinction coefficient at the respective wavelength. Furthermore, our study also reports a comparison of the analyses of the fluorescence data done with three different methods. Analyses of the fluorescence decay at 10 different fluorescence emission wavelengths revealed significant differences in both fluorescence lifetimes and the pre-exponential factor distributions. Such values differed from the values recovered from the integrated decay curves and from global analyse.

A combination of ultrasound and a bio-catalyst: removal of 2-chlorophenol from aqueous solution.

Phenolic compounds have become a cause for worldwide concern due to their persistence, toxicity and health risks. This paper reports a three-step approach to remove 2-chlorophenol from dilute aqueous solution and compares each technique. The first step utilizes Horse Radish Peroxidase (HRP) in presence of hydrogen peroxide to oxidize this organic pollutant (enzyme treatment). For a more efficient removal of 2-chlorophenol, it is necessary to add the enzyme solution gradually to the contents of the reactor instead of rapid addition. The second step, involving ultrasonic waves eliminated 2-chlorophenol through hydroxyl radical generated by the cavitation process (sono-degradation). In the third step, a combination of ultrasonic waves and enzyme was used (sono-enzyme degradation). It should be mentioned that, the enzyme can be active in the presence of ultrasonic waves under the proper sonication. The degradation has been studied at different temperatures, intensities of irradiation, and concentrations of enzyme. The rate of degradation exhibited pseudo-first order behavior and the combination method was more effective than sonolysis and enzyme treatment individually.

Modulation of the catalytic activity of free and immobilized peroxidase by extremely low frequency electromagnetic fields: dependence on frequency.

A study of the influence of electromagnetic fields (EMF) of various frequencies, from 50 up to 400 Hz, on the catalytic activity of soluble and insoluble horseradish peroxidase (POD) was carried out. To simulate the conditions in which the enzyme operates in vivo, the POD was immobilized by entrapment on a gelatin membrane or by covalent attachment on a nylon graft membrane. The rate of inactivation of the soluble POD was found to exhibit positive and negative interactions with the 1 mT applied magnetic field, with an optimum positive effect at 130 Hz. The immobilized PODs, on the contrary, do not exhibit negative interactions, but show a maximum positive interaction at 150 Hz when entrapped and at 170 Hz when covalently attached. At 50 Hz and at frequencies higher than 250 Hz no effects were observed with insoluble POD. The optimum frequency of positive interaction between the EMF and the catalytic activity of the insoluble enzymes is shifted with respect to that of the soluble enzymes towards higher frequencies, the size of the shifts being dependent on the intensity of the physical forces involved in the immobilization process.

On various possibilities in pulsed radiation biochemistry and chemistry.

Several experiments are described that relate to the application of new regimes of radiation action on enzymes in vitro and some other materials. These regimes have recently come into practice due to the appearance of a new generation of devices with very short high-energy pulses of ionizing radiation. It is shown that the term "flash radiation biochemistry" in its perfect sense has to be used at the condition of the overlapping individual effective interaction microvolumes (e.g. spurs and blobs) realized during a time interval (radiation pulse duration) that is low compared with the corresponding physical-chemical process. In this situation a number of unexpected effects occur at very low absolute doses. These processes are analyzed in terms of their non-stationary and non-diffusive developments.

Flexibility in proteins: tuning the sensitivity to O2 diffusion by varying the lifetime of a phosphorescent sensor in horseradish peroxidase.

The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656-666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. K(d) = 18 microM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O(2) to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of k(q) = 234 mm Hg(-1) s(-1) in the absence of BHA and k(q) = 28.7 mm Hg(-1) s(-1) in the presence of BHA. The quenching rate of BHA is 4000 s(-1). The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O(2) diffusion and flexibility in heme proteins.

Photochemical immobilization of proteins on microwave-synthesized photoreactive polymers.

We report a rapid and versatile procedure for the preparation of photoreactive polymers and light-induced immobilization of proteins onto such polymers. Photoreactive controlled-pore glass, silica gel, glass slide, and polystyrene microtiter plate are prepared in 40-60s by microwave irradiation of the respective amino polymers and 1-fluoro-2-nitro-4-azidobenzene. Azido group, now part of the polymer, yields highly reactive nitrene under ultraviolet (UV) light at 365 nm. Thus, when photoreactive polymer and horseradish peroxidase or glucose oxidase are exposed to UV light, the reactive nitrene immobilizes the protein molecules in 10 to 20 min through covalent bonding. As nitrene has a property of inserting into C-H bond, the method may find potential applications for immobilization of biomolecules irrespective of their functional groups.

Conformational differences between native and recombinant horseradish peroxidase revealed by tritium planigraphy.

Significant conformational differences between native and recombinant horseradish peroxidase have been shown by tritium planigraphy, which includes a method of thermal activation of tritium followed by amino acid analysis of the protein preparation. Comparison of radioactivity distribution among the amino acid residues with the theoretical (calculated) accessibility shows that the recombinant enzyme is characterized by high hydrophobicity and compactness of folding. The protective role of oligosaccharides in native enzyme has been confirmed. An unexpected result of the study is a finding on high accessibility of a catalytic histidine residue in solution. An effect of low dose (3 Gy) of irradiation on the accessibility of amino acid residues has been unequivocally demonstrated. The data can be interpreted as swelling of the compact folding and increase in the surface hydrophilicity of the recombinant enzyme. In the case of native enzyme, irradiation does not cause remarkable changes in the accessibility of amino acid residues indicating the possible extensive radical modification of the native enzyme in the life-course of the cell. The catalytic histidine is an exception. It becomes inaccessible after the enzyme irradiation, while its accessibility in the recombinant enzyme increases. An additional observation of a 5-fold decrease in the rate constant towards hydrogen peroxide points to the destructive effect of irradiation on the hydrogen bond network in the distal domain of the native enzyme molecule and partial collapse of the active site pocket.

In vitro studies of the influence of ELF electromagnetic fields on the activity of soluble and insoluble peroxidase.

The influence of an extremely low frequency (ELF) magnetic field (50 Hz and 1 mT, EMF) on the activity of a soluble and insoluble horseradish peroxidase (E.C. 1.11.17) has been studied as a function of time. Insoluble derivatives were obtained by enzyme entrapment into two different gelatin membranes or by covalent attachment of the enzyme on two nylon membranes, differently preactivated. Results have shown that the field affects the inactivation rate of the soluble enzyme, while no effects are observed with insoluble derivatives. Since in vivo enzymes are immobilised into the biomembrane bilayer or entrapped into the cytoplasmic mixture, one might speculate that our experimental conditions do not reflect the catalytic activity of the enzymes in vivo.

A method to protect sensitive molecules from a light-induced polymerizing environment.

Systems that can be polymerized in situ upon exposure to light radiation may have significant applications in tissue engineering and drug delivery. However, the light-induced polymerization step, which is the requisite for this technology, could be potentially deleterious to sensitive bioactive agents (e.g., enzymes, cytokines, matrix metalloproteinases) being entrapped. In this study, a method to protect sensitive molecules from a light-induced polymerizing environment is proposed. This method is based on the idea that nonaccessible substances cannot interact with the polymerizing species. To examine this concept, two model enzymes-namely, horseradish peroxidase and alpha-glucosidase-were protected by gelatin-based wet granulation and incorporated within a cured polyethylene glycol dimethacrylate, a photocurable monomer, under different conditions. Unprotected enzymes were used as controls. Enzymes were then allowed to diffuse out of the polymerized matrices. The activity and total enzyme recovered from these matrices by passive diffusion were compared to ascertain the extent of activity retention. Matrix assisted laser desorption ionization mass spectrometry combined with time of flight mass spectrometry (MALDI-TOF) was used to determine changes in enzyme molecular weight. During the first 24 h of diffusion from the polymerized matrices, unprotected enzymes consistently showed a loss of activity ranging from 10-66%, depending on the matrix composition and enzyme properties. In contrast, protected enzymes retained over 94% of their activity irrespective of the experimental setting. The loss of activity appears to be a direct consequence of the polymerizing environment.

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis.

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.

Energy selection is not correlated in the Qx and Qy bands of a Mg-porphyrin embedded in a protein.

The Qx-Qy splitting observed in the fluorescence excitation spectra of Mg-mesoporphyrin-IX substituted horseradish peroxidase (MgMP-HRP) and of its complex with naphthohydroxamic acid (NHA) was studied by spectral hole burning techniques. The width of a hole directly burnt in the Qy band and that of a satellite hole indirectly produced in Qy as a result of hole burning in Qx was compared. We also studied the dependence of the satellite hole in the Qy band on the burning frequency used in the Qx band. Both the directly and indirectly burnt holes were very broad in the (higher energy) Qy band. The width of the satellite hole in the Qy band was equal to the entire width of the inhomogeneously broadened band, independently from the position of hole burning in Qx. This is indicative of a clear lack of correlation between the electronic transition energies of the Qx and Qy bands. A photoproduct was produced by laser irradiation of the MgMP-HRP/NHA complex and was identified as a species with lowered Q-splitting. Conversion of the photoproduct could be achieved by thermal activation measured in temperature-cycling experiments, with a characteristic temperature of 25 K. We attribute the phototransformation to a conformational change of MgMP.

Photo-modulation of horseradish peroxidase activity via covalent attachment of carboxylated-spiropyran dyes.

The photo-modulation of the enzyme horseradish peroxidase modified with photochromic spiropyran dyes is reported. The degree of photo-modulation, greater than 90% reduction in enzyme activity under visible compared to UV illumination, matches the greatest degree of photo-modulation previously reported in the literature. The observation that only a small fraction of the conjugated photochromic molecules are photo-switched suggests that the dyes influence the enzyme activity via local interaction with the enzyme active site.

[Effect of weak combined low frequency constant and alternative magnetic fields on intrinsic fluorescence of proteins in aqueous solutions]. / Vliianie slabykh kombinirovannykh postoiannogo i peremennogo magnitnykh polei na sobstvennuiu fluirestsentsiiu riada belkov v vodnykg rastvorakh.

It was shown that weak combined static (42 microT) and low-frequency variable (40 nT; 3-5 Hz) magnetic fields change the intensity of intrinsic fluorescence of some proteins (cytochrome c, bovine serum albumin, horseradish peroxidase, alkaline phosphatase). The effect can be interpreted as a change in the conformational state of the protein in water environment by the action of weak magnetic fields. The dynamics of the process, the concentration dependence, the binding of proteins to the fluorescence probe 1,8-ANS after treatment with magnetic fields, the frequency dependence of these reactions, and the dependence of the effect on the presence of the static constituent of the magnetic field were studied. It was shown that the changes in the intrinsic fluorescence of some enzymes (horseradish peroxidase, alkaline phosphatase) are related to changes in their functional activity. It was found that the effect is partially transferred via a solvent (water, 0.01 M NaCl) preliminarily treated with magnetic field. In the solvent, changes in its intrinsic fluorescence by the action of weak magnetic fields were also registered.