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    An easy two-step purification method for human leucocyte myeloperoxidase
    (Walter De Gruyter Gmbh, 2015) Sarkarati, Bahram; Akyol, Tülay Karaağaç; Kılınç, Kamer
    Objective: The object of this study is to describe a simple, rapid and cost effective method for purification of human leucocyte myeloperoxidase from a single donor. Myelopeoxidase (MPO) was purified by a two step procedure consisting of concanavalin-A Sepharose 4B affinity chromatography followed by CM-Sephadex cation exchange chromatography. Methods: Leucocytes from a single donor collected by leucopheresis were used in purification studies. MPO was solubilized and extracted from leucocytes by homogenization in phosphate buffer containing 1% HETAB (hexadecyltrimethylammonium bromide). MPO containing soluble material was applied onto concanavalin-A Sepharose 4B affinity gel, and was eluted with methyl-a-D-manno-piranoside. Fractions with MPO activity were pooled, dialyzed and applied onto CM-sephadex cation exchange gel, and was eluted from the column at weak cationic pH with linear NaCl gradient. Results: By the use of two chromatographic procedures, MPO was purified from human leucocytes with 70% yield. Purity of MPO was checked by determining the Reinheit Zahl (RZ) value (A(430)/A(280)). The RZ value of 0.86 indicated that the purified enzyme was highly homogenous as compared to reported experimental values (ranging from 0.82 to 0.88) and pure commercial enzyme with the RZ value of 0.84. Conclusion: In comparison with earlier purification methods, the purification method reported here has higher recovery rate and high purity together. Use of leucocytes with leucopheresis origin help us to omit the leucocyte isolation step and omitting of ammonium sulphate precipitation steps also help us to reduce the cost and is shortened the time of purification.
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    A fast protein liquid chromatography method for purification of myoglobin from different species
    (2023) Sarkarati, Bahram; Milli, Nursel Söylemez; Eren, Ömer
    The aim of this study is to describe a fast method for the purification of high-purity myoglobin for Mass Spectroscopy analyses and to use it as standard-grade material. A three-step Fast Protein Liquid Chromatography (FPLC) method was used to produce high-purity myoglobin. SEC 650 gel filtration followed by an Enrich Q anion exchange chromatography was used to produce myoglobin in acceptable purity for most research methods. A second filtration step was carried out by narrow field SEC 70 gel to prepare high-purity myoglobin at standard grade purity and capable of Mass Spectroscopy analyses. At least 90% pure myoglobin was obtained by applying two chromatography steps in the samples of three species, and over 99% pure myoglobin was obtained in standard material quality and suitable for mass spectroscopy when the additional narrow field SEC 70 chromatography step was applied. The proposed method provides higher purity compared to other methods and can be applied in a shorter time. FPLC columns significantly reduce the duration of the chromatography steps. At the same time, the use of solid extraction columns instead of dialysis reduced the long overnight dialysis process to a few minutes.
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    Inhibitory effect of organic acids on human neutrophil Myeloperoxidase's peroxidation, chlorination, and nitration activities
    (Walter de Gruyter GmbH, 2023) Sarkarati, Bahram
    Objectives Myeloperoxidase from polymorphonuclear leukocytes is an important enzyme in oxidative metabolism and has a key role in tissue injuries in oxidative stress and inflammatory conditions. Therefore, its inhibitors have become the focus of studies on new drug development in recent years. The aim of the study was to determine the inhibitory effect of organic acids on the peroxidation, chlorination, and nitration activities of myeloperoxidase.Methods Seven organic acids naturally abundant in plants were tested. Different activities of myeloperoxidase were measured in the presence of various amounts of organic acids, and inhibition rates and kinetic parameters were determined for each organic acid separately.Results All the organic acids examined had inhibitory effects on the different activities of myeloperoxidase. Comparison of the IC50 values obtained for peroxidation, chlorination, and nitration activities showed that oxalic acid was the strongest inhibitor of myeloperoxidase activity, while citric acid and succinic acid were the weakest.Conclusions The results suggested that all the organic acids examined are inhibitors of myeloperoxidase. In particular, oxalic acid and fumaric acid are popular candidates for drug development research. More studies are needed to determine the in vivo effects of organic acids and their effects in the treatment of disease.
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    Nitrite oxidation and tyrosine nitration by human myeloperoxidase
    (Wiley-Blackwell, 2016) Sarkarati, Bahram; Kılınç, Kamer
    Biomineralization is deposition of hydroxyapatite, the crystallized mineral form of calcium and phosphate, by cells to their extracellular matrix (ECM), and it is an essential mechanism of bone and teeth formation in humans. Biomineralization is especially important in adults for tissue regeneration in bone defects. ECM molecules regulate mineral formation and provide crystal growth and nucleation. One of the most important ECM molecules, Alkaline Phosphatase (ALP), is the key enzyme in biomineralization process by the activity of converting organophosphate into inorganic phosphate. Moreover, osteocalcin and osteopontin are small soluble noncollagenous proteins of ECM and they regulate biomineralization by binding to calcium atoms available at crystal surfaces due to their highly negative charged amino acid residues. In this study, ALP, osteocalcin and osteopontin are expressed in bacterial systems and purified to assess in vitro biomineralization. Optimization of in vitro biomineralization activities with osteocalcin and osteopontin proteins provided the understanding of the effect of protein concentrations in crystal structure of calcium crystals. Understanding of the effect of protein concentrations will provide control over biomineralization in different cell types by designing synthetic genetic circuits. Programming non-biomineral formation cells for biomineral formation will enable differentiation free bone mineral formation. Reprogramming of non-biomineral formation cells will help to treat bone defects and bone-impairing diseases, such as osteoporosis. Consequently, it is an outstanding approach to understand the activity of bone ECM proteins and construct synthetic genetic systems that can reprogram non-mineral formation cells for biomineralization within the scope of bone tissue engineering.
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    Statins and IL-1 beta, IL-10, and MPO levels in gingival crevicular fluid: preliminary results
    (Springer/Plenum Publishers, 2016) Arı, Vuslat Çiçek; İlarslan, Yağmur Deniz; Erman, Baran; Sarkarati, Bahram; Tezcan, İlhan
    Statins possess a wide variety of pleiotropic properties that are independent of their lipid-lowering abilities such as attenuating inflammation, oxidative stress, coagulation, platelet aggregation and stimulating bone formation. The aim of the study is to evaluate the effect of statins on clinical periodontal parameters and gingival crevicular fluid (GCF) levels of IL-1 beta, IL-10, and myeloperoxidase (MPO) in inflammatory periodontal diseases. Seventy-nine subjects with hyperlipidemia and 48 systemically healthy controls (C) were included. Hyperlipidemic patients were either given a diet (HD) or prescribed statin (HS). Patients were classified into three subgroups as those who were periodontally healthy (h), who had gingivitis (g), or who had chronic periodontitis (p). Blood samples were collected for the measurement of lipid profiles. Plaque index (PI), gingival index (GI), probing pocket depth (PD), clinical attachment level (CAL), and percentage of bleeding on probing (BOP) were recorded. Gingival crevicular fluid levels of IL-1 beta, IL-10, and MPO were measured in order to determine the anti-inflammatory and antioxidant effects of statins. Probing depth values of the HSp group were significantly lower than those of the Cp group. Percentage of BOP of the HSg group was significantly lower than those of the HDg and Cg groups. While the IL-1 beta level of the HSp group was significantly lower than that of the HDp group, IL-10 levels of the HSg group were significantly higher than those of the HDg group. MPO levels were significantly lower in the HSg group when compared to those in the HDg and Cg groups. Statin use decreased the IL-1 beta and MPO levels and enhanced IL-10 in GCF. It can be suggested that statins may attenuate periodontal inflammation and progression of periodontal inflammation.