13th balkan biochemical biophysical days & meeting on metabolic disorders’ programme & abstracts
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- Burcu OKUTUCU, Ayşe DİNÇER, Figen ZİHNİOĞLU, Azmi TELEFONCU
LECTURE 2IRREGULAR IONISATION OF PROTEINS. AN ALTERNATIVE INTERPRETATION OF EXPERIMENTAL OBSERVATIONS Prof.Andrey Karshikoff Experimental measurements of ionisation equilibria of titratable groups cannot provide quantitative information for the electrostatic interactions in proteins. Moreover, in some cases an accurate prediction of electrostatic interactions is needed in order to give an adequate interpretation of the experimental observations. This is mainly due to the cooperative ionisation behaviour of the titratable groups in proteins, referred here to as irregular titration. Experimentally, irregular titration cannot be distinguished from the sum of the ionisation equilibria of more than one independent sites. This may lead to misleading interpretation of the experimental data. An example for experimental observations, which can be ambiguously interpreted, is the pH-dependence of the NMR chemical shift. The analysis of pH dependence enzymatic activity is also sensitive to how experimental data are interpreted. An illustration for such a case is the understanding of the mechanism of proton abstraction from alcohol substrate of alcohol dehydrogenase. Three hypotheses have been proposed to explain the pH-dependence of this process. All of them assume that a group from the active site of the enzyme has a pK of about 7.2 and serves of a general base for proton abstraction. Non of the groups proposed by these hypotheses have been experimentally detected. The comprehensive analysis of electrostatic interactions suggested that irregular titration of the groups in active site occurs. On the basis of this theoretical observation, a completely different molecular mechanism of the enzymatic activity of alcohol dehydrogenase can be given.
Vesna SVETLICIC and Vera ZUTIC Center for Marine and Environmental Research, Ruđer Bošković Institute, P.O.Box 180, 10002 Zagreb, Croatia svetlicic@rudjer.irb.hr Abiotic transformation of organic matter in the aquatic environments such as seawater depends more on the interfacial properties of organic matter than on its chemical composition. These properties, purely understood so far, can be measured by a direct application of the electrochemical adhesion sensor which we have developed based on our fundamental studies in the fields of surface electrochemistry and biophysics. Newly postulated biophysical processes of biopolymer selforganization into microparticles and mechanism of sol-gel phase transitions are held responsible for transformation of dissolved biopolymers and microparticles to macroscopic phases. The macroscopic gel phase appears, episodically in Northern Adriatic, as large aggregates within the water column or covering tens of square kilometres of seasurface. Current views leave no doubt on phytoplankton production and bacterial transformation of polysaccharides as main constituents of the gel matrix. The phenomenon has so far been specific for Northern Adriatic but with the global climatic changes and increasing nutrient load it could be anticipated to spread over other coastal seas of Mediterranean. There is by now accumulated evidence ranging from satellite observations to microbiological studies on sudden and dramatic changes of dispersed state (microparticles) to macroscopic gel-phase. We introduce a simple electrochemical technique to detect microparticle precursors and to follow the transition to the gel phase. When micrparticles attain critical concentration Nc (our present estimate centers around Nc 5x107 L-1) the large-scale phase transition from the dispersed to gel state takes place. AFM is introduced to image 3-D structure of the gel matrix and biopolymer molecules.
HALL A ORAL PRESENTATION 1 A SIMPLE SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF SELENIUM IN BIOLOGICAL MATERIALS Burcu OKUTUCU, Ayşe DİNÇER, Figen ZİHNİOĞLU, Azmi TELEFONCUEge University, Faculty of Science, Biochemistry Department, 35100, Bornova-İzmir/TURKEY okutucu@sci.ege.edu.tr Selenium is an essential constituent of a number of enzymes some of which have antioxidant functions. Although it is an important trace element for life, it may be toxic of only moderately higher levels of intake depending on their chemical forms. Deficiency of element in animals makes them susceptible to injury by certain types of oxidative stress correlated diseases such as; cancer, HIV infection, renal and heart disease. Selenium species in the living body can be in the form of selenoproteins, Se-containing proteins, inorganic selenium(selenite, selenate), methylated selenium and selenoamino acids. Many factors can influence the selenium contents of biological fluids and therefore values can vary in a significant way from one person to another depending on various parameters and pathological conditions. Because the selenium content of biological fluids are very low, sensitive analytical techniques are needed to measure it. Some analytical methods have been reported which the most of them require expensive instrumentation and time consuming sample preparation methods. Taking into account the characteristics of the sample and the resources available in every laboratory we tried to modify a simple and rapid spectrophotometric method for the determination of selenium in biological materials. The modified method is based on the reaction of Selenium(Se IV) with potassium iodide in acidic medium to liberate iodine which the absorbance decrease is directly proportional to selenium concentration by the use of thionin dye Calibration graph was maintained at 0.02-0.3 g range in a total volume of 2 ml. For the preparation of plasma samples direct dilution and enzymatic digestion methods were performed and compared. Variation coefficients and standard deviations were calculated for five replicate determinations. Furthermore, as well as the accuracy, application of the method to the biological materials was investigated by using a commercial drug (Dietary Selenium Supplement) and plasma samples. Selenium in water, soil, plant materials, cosmetics, etc. can be also determined by this method.
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