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.

Denaturation of the SARS-CoV-2 spike protein under non-thermal microwave radiation.

SARS-CoV-2, the virus that causes COVID-19, is still a widespread threat to society. The spike protein of this virus facilitates viral entry into the host cell. Here, the denaturation of the S1 subunit of this spike protein by 2.45 GHz electromagnetic radiation was studied quantitatively. The study only pertains to the pure electromagnetic effects by eliminating the bulk heating effect of the microwave radiation in an innovative setup that is capable of controlling the temperature of the sample at any desired intensity of the electromagnetic field. This study was performed at the internal human body temperature, 37 °C, for a relatively short amount of time under a high-power electromagnetic field. The results showed that irradiating the protein with a 700 W, 2.45 GHz electromagnetic field for 2 min can denature the protein to around 95%. In comparison, this is comparable to thermal denaturation at 75 °C for 40 min. Electromagnetic denaturation of the proteins of the virus may open doors to potential therapeutic or sanitation applications.

Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays-Protein and Buffer Composition Contribution.

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.

Free Radicals and ROS Induce Protein Denaturation by UV Photostability Assay.

Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180-250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.

The cataract-related S39C variant increases γS-crystallin sensitivity to environmental stress by destroying the intermolecular disulfide cross-links.

γS-crystallin, a crucial structural lens protein, plays an important role in maintaining lens transparency through its solubility and stability. The S39C mutation, a proven pathogenic mutation involved in congenital cataract, resulted in progressive cataract in adolescents. In this study, using biophysical methods, we thoroughly investigated the effects of the S39C mutation on the γS-crystallin structure, stability and propensity for aggregations. The data from spectroscopy analyses did not reveal an effect of the S39C mutation on the native structure of monomeric γS-crystallin. However, when faced with oxidative conditions, the S39C mutation prevented γS-crystallin from forming stable disulfide-linked dimers and remarkably increased hydrophobicity and the propensity to aggregate and precipitate. Under UV irradiation, heat shock, and GdnHCl-induced denaturation, the S39C mutant tended to aggregate and was prone to form more deleterious aggregates than the wild type protein. Therefore, the S39C mutation significantly increased the sensitivity of γS-crystallin to environmental stress. However, the addition of αA-crystallin and lanosterol did not change the tendency of the mutant to aggregate. According to molecular dynamic (MD) simulations, the S39C mutation had little effect on the secondary or tertiary structures of monomeric γS-crystallin but disrupted the disulfide-linked structure of the γS-crystallin dimer. The cleavage of this bond might largely reduce the structural stability of γS-crystallin. The significant decrease in the structural stability along with the increasing aggregation tendency under environmental stress might be the major causes of progressive juvenile onset cataracts induced by the S39C mutation.

Radiofrequency therapy in esthetic dermatology: A review of clinical evidences.

BACKGROUND: Chronological skin aging causes the modification of genetic material through enzymes and proteins changes. The process reduces cellular proliferation, along with loss of tissue elasticity, reduced ability to regulate aqueous exchanges, and inefficient tissue replication. Appearance is negatively affected by cumulative changes in coloration, texture, and elasticity over time. The increase in the population's average life expectancy boosts the search for cosmetic therapies that can delay aging, mostly for the noninvasive modalities. Among the various options, radiofrequency therapy is a technique that can help reduce the effects of skin aging. AIM: Therefore, this study aims to review clinical evidence provided by scientific literature on the benefits of using radiofrequency therapy in reducing skin aging effects. METHODS: A review of the literature concerning skin aging, characteristics of radiofrequency therapy, and radiofrequency therapy in the treatment of skin laxity and mechanism of action was conducted using PubMed. RESULTS: The included studies have suggested that the mechanism of radiofrequency action is heating the dermis while preserving the epidermis. This heating causes immediate collagen denaturation, which is followed by the formation of new collagen, naturally providing skin tightening and greater elasticity. CONCLUSION: Even when used as single therapeutic modality, radiofrequency seems to meet the expectations in reducing the effects of skin aging.

Denaturation of Lysozyme with Visible-light-responsive Photocatalysts of Ground Rhodium-doped and Ground Rhodium-antimony-co-doped Strontium Titanate.

Protein denaturants play an important role in medical and biological research, and development of new denaturants is widely explored to study aging and various diseases. In this research, we treated lysozyme, a model protein, with photocatalysts of ground Rh-doped SrTiO3 (g-STO:Rh) and ground Rh-Sb-co-doped SrTiO3 (g-STO:Rh/Sb) under visible light irradiation to explore the potential of those photocatalysts as denaturants. SDS-PAGE showed that photocatalysis with g-STO:Rh induced the fragmentation of lysozyme into unidentifiable decomposition products. BCA and Bradford protein assays indicated that the peptide bonds and basic, aromatic and N-terminal amino acid residues in lysozyme were denaturated by g-STO:Rh photocatalysis. The denaturation of those amino acids, as quantified by the decreased solubility of lysozyme, was estimated to be more severe by Bradford protein assay than by BCA protein assay. Circular dichroism (CD) spectra of lysozyme revealed that the secondary structure was denatured by g-STO:Rh photocatalysis, indicating that g-STO:Rh photocatalysis is especially effective against the amino acid residues that form the secondary structure via hydrogen bonds. Furthermore, the lytic activity of lysozyme was reduced by g-STO:Rh photocatalysis, owing to denaturation of the enzyme. The visible-light-responsive photocatalyst of g-STO:Rh/Sb accelerates the oxidation reaction and has stronger oxidizing power than g-STO:Rh. Lysozyme was denatured more quickly by g-STO:Rh/Sb photocatalysis than by g-STO:Rh according to analysis by SDS-PAGE, CD spectroscopy, BCA and Bradford protein assays, and lytic activity. These results suggest that higher photocatalytic activity induces more significant denaturation of lysozyme, implying that the main factor of photocatalytic denaturation of lysozyme is oxidation. It should be noted that, as far as we know, this is the first report for denaturation of protein using visible-light-responsive photocatalyst.

Light-Induced Covalent Buffer Adducts to Histidine in a Model Protein.

PURPOSE: Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His. METHODS: A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase. RESULTS: The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking. CONCLUSIONS: In nucleophilic buffers, light can induce covalently-linked adducts to His residues.

The protective action of osmolytes on the deleterious effects of gamma rays and atmospheric pressure plasma on protein conformational changes.

Both gamma rays and atmospheric pressure plasma are known to have anticancer properties. While their mechanism actions are still not clear, in some contexts they work in similar manner, while in other contexts they work differently. So to understand these relationships, we have studied Myoglobin protein after the treatment of gamma rays and dielectric barrier discharge (DBD) plasma, and analyzed the changes in thermodynamic properties and changes in the secondary structure of protein after both treatments. The thermodynamic properties were analyzed using chemical and thermal denaturation after both treatments. We have also studied the action of gamma rays and DBD plasma on myoglobin in the presence of osmolytes, such as sorbitol and trehalose. For deep understanding of the action of gamma rays and DBD plasma, we have analyzed the reactive species generated by them in buffer at all treatment conditions. Finally, we have used molecular dynamic simulation to understand the hydrogen peroxide action on myoglobin with or without osmolytes, to gain deeper insight into how the osmolytes can protect the protein structure from the reactive species generated by gamma rays and DBD plasma.

Elucidation of µs dynamics of domain-III of human serum albumin during the chemical and thermal unfolding: A fluorescence correlation spectroscopic investigation.

The local structural dynamics and denaturation profile of domain-III of HSA against guanidine hydrochloride (GnHCl) and temperature has been studied using a coumarin based solvatochromic fluorescent probe p-nitrophenyl coumarin ester (NPCE), covalently tagged to Tyr-411 residue. By the steady state, time-resolved and single molecular level fluorescence studies it has been established that the domain-III of HSA is very sensitive to GnHCl but somewhat resistant to temperature and the domain specific unfolding proceeds in an altered way as compared to the overall unfolding of HSA. While the overall denaturation of HSA is a two-state process for both GnHCl and heat, domain-III adopts two intermediate states for GnHCl induced denaturation and one intermediate state for temperature induced denaturation. Fluorescence correlation spectroscopic investigation divulges the conformational dynamics of domain-III of HSA in the native, intermediates and denatured state.

Changes in nail keratin observed by Raman spectroscopy after Nd:YAG laser treatment.

Lasers and photodynamic therapy have been considered a convergence treatment for onychomycosis, which is a fungal infection on the nail bed and nail plate. Laser therapies have shown satisfactory results without significant complications for onychomycosis; however, the mechanism of clearing remains unknown. In this work, we investigated changes in the chemical structure of nail keratin induced by Nd:YAG laser using Raman spectroscopy. Toe nails with onychomycosis were treated with 1064 nm Nd:YAG laser. After laser treatment, the disulfide band (490-590 cm-1 ) of nail keratin was rarely observed or was reduced in intensity. The amide I band (1500-1700 cm-1 ) also showed changes induced by the laser. The α-helical (1652 cm-1 ) structures dominated the ß-sheet (1673 cm-1 ) in nontreated nail, but the opposite phenomenon was observed after laser treatment.

Gold nanoparticles increases UV and thermal stability of human serum albumin.

Ultraviolet (UV) radiation, temperature, and time can degrade proteins. Here, the authors show that gold nanoparticles significantly protect human serum albumin from denaturation when exposed to "stressing" conditions such as UV irradiation and sustained exposure in suboptimal conditions. In particular, the authors show that gold nanoparticles significantly reduce the decrease in secondary structure induced by UV irradiation or extended exposure to ambient temperature.

Theoretical and practical aspects relating to the photothermal therapy of tumors of the retina and choroid: A review.

Photothermal treatment of tumors of the retina and choroid such as retinoblastomas, malignant melanomas, benign tumors as well as of vascular malformations can be performed by using laser radiation. A number of basic physical laws have to be taken into account in this procedure. Of particular importance thereby are: Arrhenius' law to approximate the kinetics of protein denaturation and photocoagulation, furthermore the electromagnetic radiation field, the distribution of both radiant and thermal energy induced in tumors and vascular structures, the influence of the wavelength and laser pulse duration (exposure time), as well as of the optical properties of the tissue. Strict confinement of the extent of the photothermal damage is critical since such pathological entities are frequently located close to the macula or optic nerve head.The conditions for tumor destruction are best fulfilled when using radiation in the near-infrared range of the electromagnetic spectrum such as that emitted from the diode (810 nm) and the Nd: YAG (1064 nm) laser, because of the good optical penetration properties of these radiations in tissue. Short wavelength sources of radiation, such as the argon ion (488, 514 nm) or the freqeuency-doubled Nd: YAG (532 nm) laser are less well suited for the irradiation of large vascular structures due to their poor penetration depths. However, for vascular formations with a small thickness (1 mm or less), short wavelength sources appear to be the most appropriate choice. Optical coupling of radiant energy to the eye by means of indirect ophthalmoscopic systems or positive contact lenses is furthermore of importance. Strong positive lenses may lead to severe constrictions of the laser beam within the anterior segment, that leads to high irradiance increasing the probability for structures to be damaged; with negative contact lenses, such as the -64 D Goldmann type lens, this danger is largely absent.

Expression and characterization of a cold-adapted, thermotolerant and denaturant-stable GH5 endoglucanase Celal_2753 that withstands boiling from the psychrophilic bacterium Cellulophaga algicola IC166(T).

OBJECTIVES: To characterize a novel endoglucanase, Celal_2753, from the psychrophilic bacterium Cellulophaga algicola IC166(T). RESULTS: Celal_2753 was purified to homogeneity with a yield of 81 % and with a molecular weight of 40 kDa on SDS-PAGE. It had maximum hydrolytic activity towards carboxymethyl cellulose at 40 °C and pH 6. It showed 33 % of the maximum activity at 10 ºC. Its activity increased to 272-316 % in the presence of 0.25-2 M NaCl and KCl at 40 °C. Celal_2753 was stable in the presence of 10 % (v/v) Tween 20, 10 % (v/v) Triton X-100, 16 mM SDS, 6 M urea or 2 M guanidine hydrochloride. Celal_2753 that had been boiled for 5 min recovered 55 % of its initial activity by incubating at 30 °C for 60 min. CONCLUSION: Because of its cold-adapted, thermotolerant and denaturant-stable properties, endoglucanase Celal_2753 is promising in detergent industry and bioethanol production.

Stabilizing effect of biochar on soil extracellular enzymes after a denaturing stress.

Stabilizing extracellular enzymes may maintain enzymatic activity while protecting enzymes from proteolysis and denaturation. A study determined whether a fast pyrolysis hardwood biochar (CQuest™) would reduce evaporative losses, subsequently stabilizing soil extracellular enzymes and prohibiting potential enzymatic activity loss following a denaturing stress (microwaving). Soil was incubated in the presence of biochar (0%, 1%, 2%, 5%, or 10% by wt.) for 36 days and then exposed to microwave energies (0, 400, 800, 1600, or 3200 J g(-1) soil). Soil enzymes (ß-glucosidase, ß-d-cellobiosidase, N-acetyl-ß-glucosaminidase, phosphatase, leucine aminopeptidase, ß-xylosidase) were analyzed by fluorescence-based assays. Biochar amendment reduced leucine aminopeptidase and ß-xylosidase potential activity after the incubation period and prior to stress exposure. The 10% biochar rate reduced soil water loss at the lowest stress level (400 J microwave energy g(-1) soil). Enzyme stabilization was demonstrated for ß-xylosidase; intermediate biochar application rates prevented a complete loss of this enzyme's potential activity after soil was exposed to 400 (1% biochar treatment) or 1600 (5% biochar treatment) J microwave energy g(-1) soil. Remaining enzyme potential activities were not affected by biochar, and activities decreased with increasing stress levels. We concluded that biochar has the potential to reduce evaporative soil water losses and stabilize certain extracellular enzymes where activity is maintained after a denaturing stress; this effect was biochar rate and enzyme dependent. While biochar may reduce the potential activity of certain soil extracellular enzymes, this phenomenon was not universal as the majority of enzymes assayed in this study were unaffected by exposure to biochar.

Effects of green and red light in ßL-crystallin and ovalbumin.

The effects of visible light on biological systems have been widely studied. In particular, the alterations of blue light on the ocular lens have recently attracted much attention. Here, we present a study about the effects produced by green and red light on two different proteins: ßL-crystallin and ovalbumin. Based on differential scanning calorimetry (DSC), circular dichroism (CD), dynamic light scattering (DLS), and fluorescence emission measurements, we found that both wavelengths induce structural changes in these proteins. We also observed that ßL-crystallin aggregates. Our work may advance our understanding about conformational and aggregation processes in proteins subjected to visible radiation and the possible relationship with cataracts. While blue light has been considered the only harmful component in the visible espectrum, our findings show the possibility that lower energy components may be also of some concern.

Cryoprotective ability of betaine-type metabolite analogs during freezing denaturation of enzymes.

OBJECTIVE: To evaluate an analog library of betaine-type cellular metabolites, which are naturally found in polar fish for survival in subzero temperatures, for preventing denaturation of enzymes during freezing. RESULTS: Comparison of the cryoprotective ability of reported cryoprotectants, such as dimethylsulfoxide, glycerol, ectoine, hydroxyectoine, and trehalose, with betaine-type analogs using α-glucosidase revealed that analogs introducing C3-C6 alkyl chains into an ammonium cation retained 20 % higher activity than the control cryoprotectants at the same concentration. In particular, the analog possessing triplicate n-butyl chains showed a profound effect. It allowed retention of enzyme activity to 95 % even after 100 freeze-thaw cycles, while addition of the control cryoprotectants decreased the activity to 10-20 %. The cryoprotective ability of betaine-type analogs can be applied not only to α-glucosidase but also other enzymes such as ß-glucosidase, alkaline phosphatase, lactose dehydrogenase, sulfatase, and horseradish peroxidase. CONCLUSION: Synthetic betaine-type metabolite analogs possess practicable cryoprotective ability for various enzymes, and are considerably superior to previously reported cryoprotectants.

Quantitative analysis of the thermal stability of the gamma phage endolysin PlyG: a biophysical and kinetic approach to assaying therapeutic potential.

Endolysins are lytic enzymes encoded by bacteriophage that represent an emerging class of protein therapeutics. Considering macromolecular thermoresistance correlates with shelf life, PlyG, a Bacillus anthracis endolysin, was thermally characterized to further evaluate its therapeutic potential. Results from a biophysical thermal analysis revealed full-length PlyG and its isolated domains comprised thermal denaturation temperatures exceeding 63°C. In the absence of reducing agent, PlyG was determined to be kinetically unstable, a finding hypothesized to be attributable to the chemical oxidation of cysteine and/or methionine residues. The presence of reducing agent kinetically stabilized the endolysin, with PlyG retaining at least ~50% residual lytic activity after being heated at temperatures up to 80°C and remaining enzymatically functional after being boiled. Furthermore, the endolysin had a kinetic half-life at 50°C and 55°C of 35 and 5.5h, respectively. PlyG represents a thermostable proteinaceous antibacterial with subsequent prolonged therapeutic shelf life expectancy.

[Degradation of Mitochondria to Lipofuscin upon Heating and Illumination].

In the present work, it has been shown that the isolated mitochondria can undergo transformation to lipofuscin granules without any additional factors (oxygen saturation, prooxidants). The process occurs spontaneously and slowly at low temperature, and rapidly--by heating (thermo-lipofuscin) or under UV irradiation (photo-lipofuscin). The main contribution to the formation of mitochondrial lipofuscin comes from denatured proteins. Thermo-formation of lipofuscin depends on lipid peroxidation, while the presence of lipids is not required for photo-lipofuscin formation. It is shown that the use of detergent able to degrade mitochondria is necessary to measure lipofuscin content properly.

Assessing of plasma protein denaturation induced by exposure to cadmium, electromagnetic fields and their combined actions on rat.

In our environment, we have numerous chances to be exposed to not only electromagnetic fields (EMFs) but also many chemicals containing mutagens. Therefore, the aim of this study was to estimate whether rat's exposure to cadmium and/or EMFs could cause oxidative damage to molecular structure of proteins and whether and to what extent the effects of co-exposure differ from those observed under the treatment with each exposure alone. Thirty-two rats were divided into four groups. Group 1 was termed as control, group 2 was treated with cadmium (3.0 mg/Kg), group 3 was exposed to EMF (10 mT/h/day) and group 4 was treated with cadmium and exposed to EMF. Protein carbonyls (PCO) in the plasma as a marker of oxidative protein damage and total oxidant status (TOS), as well as electrical conductivity and SDS electrophoresis to estimate changes in molecular structure of protein, were determined. The exposure to Cd and/or EMF led to oxidative protein damage (increased PCO and TOS) accomplished by increased stress of electrical charges on the surface of the protein molecule (increased electrical conductivity) and changes in the molecular structure of protein. The effects were more pronounced after treatment with both Cd and EMF than at the treatment with each exposure alone. The serious damage to proteins at the co-exposure to Cd and EMF seems to be due to the interference of the EMF with the toxic activity of cadmium. This work concluded that combined exposure to Cd and EMFs might increase the risk of plasma damage via enhancing free radical generation and protein oxidation.