Kinetic changes in activity of HR-peroxidase, induced by very low doses of phenol

The allosteric protein of horseradish peroxidase (HRP) shows two main types of activity, peroxidase and oxidase, depending on the kind of low molecular effectors. The effects of very low doses of phenol, prepared by successive dilutions in water or in 75% ethanol, on initial HRP activity in oxidation of o-dianisidine or luminol were tested in a systematic manner by colorimetric and luminometric methods. Results showed that phenol dilutions, including those below Avogadro’s number, could activate or inhibit HRP in peroxidase and oxidase type reactions with a sinusoidal pattern. Km values for the studied substrates changed parallel to HRP peroxidase/oxidase activity and the maximum activity in the peroxidase reaction corresponded to the minimum activity in the oxidase reaction and vice versa. The effect also depended on the type of dilutor. The observations of the peroxidase/oxidase oscillations in the sinusoidal pattern of HRP activity, dependent on the rate of phenol dissolution and the time of preincubation, point out to the conclusion that HRP might be a good model for high dilutions research. The experiments provide strong evidence that horseradish peroxidase (HRP) is a very sensitive detector of subtle changes in the concentration of phenol used as a cofactor in the peroxidase/oxidase reaction. Keywords: HR-peroxidase, peroxidase-oxidase, phenol, hormesis, homeopathy, high dilutions.  Mudanças cinéticas na atividade da HR-peroxidade induzidas por doses muito baixas de fenol Resumo A proteína alostérica da peroxidase do rabano (HRP) mostra dois tipos principais de atividade, peroxidase e oxidase, de acordo com o tipo de efetores de baixa molecularidade. Os efeitos de doses muito baixas de fenol, preparadas através de diluições sucessivas em água ou etanol 75% na atividade inicial da HRP sobre a oxidação da o-dianisidina ou luminol for testados de modo sistemático através de métodos colorimétricos e luminométricos. Os resultados mostram que as diluições de fenol, incluindo aquelas por baixo do número de Avogadro, foram capazes de ativar ou inibir a HRP em reações de tipo peroxidade e oxidase com um padrão sinusoidal. os valores Km dos substratos estudados variaram paralelamente àatividade peroxidase/oxidase da HRP; a atividade máxima da reação peroxidase correspondeu àatividade mínima na reação oxidase e vice-versa. O efeito também se mostrou dependente do tipo do solvente. A observação das oscilações sinusoidais na atividade da HRP, dependentes da taxa de dissolução do fenol e do tempo de pré-incubação, permitem concluir que a HRP pode ser um bom modelo na pesquisa das altas diluições. Os experimentos oferecem fortes evidéncias a favor da HRP como detector muito sensível de mudanças mínimas na concentração do fenol, utilizado como cofator na reação peroxidase/oxigenase. Palavras-chave: HR-peroxidase, peroxidase-oxidase, fenol, hormese, homeopatia, altas diluições.  Cambios cinéticos en la actividad de la HR-peroxidasa inducidos por dosis muy bajas de fenol Resumen La proteína alostérica de la peroxidasa del rábano (HRP) muestra dos tipos principales de actividad, peroxidasa y oxidasa, dependiendo del tipo de efectores de baja molecularidade. Los efectos de doses muy bajas de fenol, preparadas mediante diluciones sucesivas en agua o etanol al 75% sobre la actividad inicial de la HRP sobre la oxidación de o-dianisidina o luminol fueron testados de modo sistemático mediante métodos colorimétricos y luminométricos. Los resultados muestran que las diluciones de fenol, incluyendo aquellas abajo del número de Avogadro, pudieron activar o inhibir la HRP en reacciones de tipo peroxidasa y oxidasa con un patrón sinusoidal. Los valores Km de los sustratos estudiados variaron paralelamente a la actividad peroxidasa/oxidasa de la HRP; la actividad máxima de la reacción peroxidasa correspondió a la actividad mínima en la reacción oxidasa y viceversa. El efecto también se mostró dependiente del tipo de solvente. La observación de las oscilaciones sinusoidales en la actividad de la HRP, dependientes de la tasa de disolución del fenol y del tiempo de preincubación, llevan a concluir que la HRP puede ser un buen modelo para la investigación de las altas diluciones. Los experimentos ofrecen fuertes evidencias a favor de la HRP como detector muy sensible de cambios mínimos en la concentración de fenol, utilizado como cofactor en la reacción peroxidasa/oxigenasa. Palabras-clave: HR-peroxidasa, oxidasa-peroxidasa, fenol, hormesis, homeopatía, altas diluciones.  Correspondence author: Elzbieta Malarczyk, [email protected] How to cite this article: Malarczyk E. Kinetic changes in the activity of HR-peroxidase induced by very low doses of phenol. Int J High Dilution Res [online]. 2008 [cited YYYY Mmm DD]; 7(23): 48-55. Available from: http://journal.giri-society.org/index.php/ijhdr/article/view/37/349. ÂÂÂ

Being attendant at pediatric clinics can increase bacterial contamination risk on hands?

Objective: The aim of this study was to examine the bacterial growth of the attendants’ hands and to determine the difference between the hospitalized and non-hospitalized attendants’ hands’ bacterial growth. Materials and Methods: In this study, the samples taken from the hands of attendants who were accompanying the hospitalized patients were examined. As a control group attendants who were not hospitalized was chosen. The samples were taken from the attendants’ hands and between the fingers using the swap which was wetted by steril serum physiological. Samples were inoculated to bood agar (BA) and eosine methylene blue agar (EMB) immediately. Inoculated plates were incubated 24-48 hours at 370C and growth was evaluated. Growth was identified using colony morphology, Gram stain, catalase and oxidase reaction. Catalaz positive bacteria were investigated for beta hemolysis, colony morphology on BA, Gram stain and microscopic appearance. Growth characteristics of Gram-negative bacilli on EMB and microscopic appearance were determined. All samples were identified by Vitec II. Results: All participants were women and their average age were 28.93±6.09 (min=18, max=45). It was determined that the rate of bacterial growth was 10% in case group and there was not growth in control group. The difference was statistically insignificant (χ² = 5.263, p = 0.056). Conclusions: In conclusion, the bacterial growth on 10% of participants’ hands in case group is an important and remarkable result. Being at the border of statistical difference between the two groups suggest that similar studies should be conducted with a larger sample group. Bangladesh Journal of Medical Science Vol.20(2) 2021 p.263-267

Complexity of a Peroxidase-Oxidase Reaction Model

The peroxidase-oxidase oscillating reaction was the first (bio)chemical reaction to show chaotic behaviour. The reaction is rich in bifurcation scenarios, from period-doubling to peak-adding mixed mode oscillations. Here, we study...

Allosteric cooperation in a de novo-designed two-domain protein

We describe the de novo design of an allosterically regulated protein, which comprises two tightly coupled domains. One domain is based on the DF (Due Ferri in Italian or two-iron in English) family of de novo proteins, which have a diiron cofactor that catalyzes a phenol oxidase reaction, while the second domain is based on PS1 (Porphyrin-binding Sequence), which binds a synthetic Zn-porphyrin (ZnP). The binding of ZnP to the original PS1 protein induces changes in structure and dynamics, which we expected to influence the catalytic rate of a fused DF domain when appropriately coupled. Both DF and PS1 are four-helix bundles, but they have distinct bundle architectures. To achieve tight coupling between the domains, they were connected by four helical linkers using a computational method to discover the most designable connections capable of spanning the two architectures. The resulting protein, DFP1 (Due Ferri Porphyrin), bound the two cofactors in the expected manner. The crystal structure of fully reconstituted DFP1 was also in excellent agreement with the design, and it showed the ZnP cofactor bound over 12 Å from the dimetal center. Next, a substrate-binding cleft leading to the diiron center was introduced into DFP1. The resulting protein acts as an allosterically modulated phenol oxidase. Its Michaelis–Menten parameters were strongly affected by the binding of ZnP, resulting in a fourfold tighter Km and a 7-fold decrease in kcat. These studies establish the feasibility of designing allosterically regulated catalytic proteins, entirely from scratch.

Detection of Aeromonas hydrophila in Raw Milk and Soft Cheese in Baghdad City

The frequency and distribution pattern of Aeromonas hydrophilain cow’s raw milk and soft cheese were investigated in Abu-Ghraib, Al-Fudhaliyah and Al-Sadrya regions in Baghdad. A total of one hundred twenty pooled samples were collected during February until June (2019),in which sixty samples of raw milk pooled from milk containers, twenty from each region (four samples collected monthly per region) and sixty samples of fresh soft cheese made from raw unpasteurized milk were collected from different supermarkets in Baghdad as same as proportion above. Modified procedures for isolation and identification were dependent in which, modified tryptone soya yeast extract sheep blood agar supplemented with ampicillin used. Gram stain and oxidase reaction aid in segregation. Modified Congo red assay used to detect biofilm and Kirby-Bauer disc diffusion method was used for determination sensitivity of isolates toampicillin (AM 10µg),cephalexin (CLX, 30µg), azithromycin (AZM, 15µ) andvancomycin (VA 30µg). The results confirmed recovery of eleventh isolates (9.16 %) from one hundred twenty pooled samples, in which four isolates (3.33 %) from raw milk samples; Two (1.66 %) from Abu-Ghraib and one(0.83 %) from each other regions. Seven isolates (5.83 %) were detected from fresh soft cheese samples in which, three (2.5 %) from Abu-Ghraib and two (1.66 %) from each other regions. All isolates were Gram-negative rod- shaped, oxidase positive and biofilm producers. Four isolates (36.36 %) were resistant to selected antibiotics in which, two (18.18 %) from Abu-Ghraib: One (9.09 %) from raw milk and soft cheese; and one (9.09 %) in each other regions in fresh soft cheese only. In conclusions. Milk and cheese production in Abu-Ghraib, Al-Fudhaliyah and Al-Sadrya regions in Baghdad are encountered by the problems of bacterial contamination and presence of multidrug resist strain of A. hydrophila which considered a risk to public health, may be due both insufficient and misuse treatment with antibiotics or in somewhat due to bad quality and/or poor hygiene of processing involved in milk production. Thus recommended monitoring these products for better hygienic status

First report of citrus bacterial canker caused by Xanthomonas axonopodis pv. citri in iraq

Collected citrus leaves, with erumpent, callus – like lesions with a water-soaked margin of Tangerine (Citrus reticulate Blance), Mexican lemon (Citrus limon), and Sweet orang (Citrus sinensis Osbec) from different orchards in Diyala, Baghdad and Babylon Governorate of Iraq. Pathogenicity of different isolates was confirmed along with symptoms under laboratory condition on detached leaf using pin prick method, observed that 21 (60 %) isolate Xac. was highly virulent to initiate water soaked lesion and fully developed symptoms within 10 to15 days, 14 (40 %) isolate Xac. were less virulent. The results of morphological (shape, colony and color) and biochemical characteristic (gram reaction, starch hydrolysis, gelatein liquefaction, KOH test, Indole production, oxidase reaction, acid and gas production, catalase reaction, fluorescent pigmentation test and NaCl tolerant). Molecular identification was performed by PCR using specific primer J-pth1̸ J-pth2 amplicon 198 bp indicated the occurrence of CBC in Iraq. All isolates 35(100%) were harbor pthA gene. These represent the first report of CBC in Iraq.

Identification of a seed-borne rice bacterium, Burkholderia glumae using cultural, morphological and biochemical methods

Burkholderia glumae is a seed-borne rice bacterium that causes bacterial panicle blight (BPB), which is a promising disease in many rice-growing areas around the world. The bacterium has been successfully grown on King’s B agar medium (KBM) at 280C for 48 hrs. It is Gram negative, non-spore-forming, rod-shaped with cell size 0.5 to 0.7 - 1.5 to 2.0 μm with rounded ends. The colony appears as grayish white or yellow due to the pigment. The bacterium gave positive test in gelatin liquefaction, KOH solubility and nitrate reduction and while negative test in starch hydrolysis, oxidase reaction, levan production and arginine dihydrolase test. The bacterium utilized different carbon sources viz., sugars, amino acids, sugar alcohols and organic acids when examined through BiologTMGN2 Microplate System. This study would help in control and management of seed-borne bacterial plant pathogen B. glumae.

Mitochondrial cytochrome c oxidase: mechanism of action and role in regulating oxidative phosphorylation

Mitochondrial oxidative phosphorylation has a central role in eukaryotic metabolism, providing the energy (ATP) required for survival. Regulation of this important pathway is, however, still not understood, largely due to limitations in the ability to measure the essential metabolites, including oxygen (pO2, oxygen pressure), ADP, and AMP. In addition, neither the mechanism of oxygen reduction by mitochondrial cytochrome c oxidase nor how its rate is controlled is understood, although this enzyme determines the rate of oxygen consumption and thereby the rate of ATP synthesis. Cytochrome c oxidase is responsible for reduction of molecular oxygen to water using reducing equivalents donated by cytochrome c and for site 3 energy coupling in oxidative phosphorylation. A mechanism-based model of the cytochrome c oxidase reaction is presented in which transfer of reducing equivalents from the lower- to the higher-potential region of the coupling site occurs against an opposing energy barrier, Q. The steady-state rate equation is fitted to data for the dependence of mitochondrial respiratory rate on cytochrome c reduction, oxygen pressure (pO2), and [ATP]/[ADP][Pi] at pH 6.5 to 8.35 (where Pi is inorganic phosphate). The fit of the rate expression to the experimental data is very good for all experimental conditions. Levels of the intermediates in oxygen reduction in the oxidase reaction site have been calculated. An intermediate in the reaction, tentatively identified as peroxide, bridged between the iron and copper atoms of the reaction site has a central role in coupling mitochondrial respiration to the [ATP]/[ADP][Pi].

Kinetic study of glucose oxidase on microfluidic toner-based analytical devices for clinical diagnostics with image-based detection

This report describes a kinetic study for the glucose oxidase reaction on microfluidic toner-based analytical devices based on digital image analysis.