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Analysis of in vitro experiments Dr. Kruglova E.B.

What is a concentration dependence in Molecular Biophysics?.

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Analysis of in vitro experiments Dr. Kruglova E.B.

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  1. What is a concentration dependence in Molecular Biophysics? Practically in every experimental method that is used to study binding of the small molecules (ligands) to biomacromolecules (DNA's, RNA's, oligonucleotides, proteins) some dependencies between recorded values and concentrations of reagents are observed. In the lecture, I want to describe generally the ways and the models, which we can use to determine the binding parameters in a ligand-biopolymer system from analysis of these dependencies: affinity. stoichiometric coefficients, site sizes to chose the best binding modes. Analysis of in vitro experiments Dr. Kruglova E.B.

  2. Why do we still continue - to melt NA (DNA) at temperatures more than 100 0C ? to add in solutions of biopolymers NaCl, KCl in such quantity to be more than 1 M and ions of two charged and transition metals? to expose of solutions of biomacromolecules to higher doses either ionizing radiation or drugs? If our living conditions are 37 0C, about 0.1 M NaCl and pH7.

  3. So obtainig results in these in vitro experiments can be fine and very simple Models describing different situation in living cells and organisms in both real and critical situation. Thus we must continue to work in the same way

  4. What is a concentration dependence? Solutions of the components A and B Our conclusion: component A interacts with component B – Why? Colour is changed to A is added B CA0 CB0 A (ligand) can be a small molecule of dyes, drugs, aminoacids, proteins, so on B can be in molecular biophysics NA's, proteins, oligonucleotides so on If that ibformation is not enougth

  5. What is a concentration dependence? Spectrofluorometer Spectrophotometer CD spectropolarimeter so on

  6. What is a concentration dependence? Spectrofluorometer Spectrophotometer, VIS range Can we say that the component A interacts with component B using only one spectra? - No. We shoul add to spectra of the mixture A+B, at least, spectra of free component A at the same concentration.

  7. Thus, good concentration dependencies we can obtain by titration procedure A titration is a method of analysis that will allow to determine the precise endpoint of a reaction and therefore the precise quantity of reactant in the titration ..(Wikipedia). There are two basic ways that are used usually in biophysics experiments Case 1 The method can give very good results for studies of interaction between dyes or drugs and NA's and proteins Why? V1 ofCA0 is added to V2 of (CA0 +CB0 ) can be shown that at that titration procedure concnetration of component A does not change in each (A+B) mixture and to be CA0 = (CA0 V1 + V2 CA0) /(V1+ V2 ) CA0 CA0 +CB0

  8. How can we obtain the thermodynamic and spectral parameters of a drug to DNA binding by spectrophotometry? CAoi = [Cf ]i + [Cb ]i CL0 = If one type of complex is formed only If omponent B does not absorb in VIS range Ai = f [Cf ]i + b [Cb ]i

  9. Scatchard isotherm (plot) r/Cf Then we can construct Scatchard plot without any preliminary knowledge about a mode of binding A to B and obtain values of binding constant and site size as it is shown in the picture K [Cf] is equilibrium concentration of a free ligand r = [Cb ]/CBO r n=1/r

  10. It is very difficult to obtain good concentration dependence in biophysical expeiment, which we can analysis to obtain the thermodynamic parametrs of interaction A+B. Why? Very often in biochemical experiments it is used complicate buffer systems consisting of some organical components (TRIS, EDTA), two carged metalloions, so on; At interaction of many biopolymers with some dyes, drugs, metal ions heving more than one ionic charge aggregation, sedimentation of biomacromolecules or condensation of DNA's (NA) are observed; Large organic molecules which we study as ligands can form dimers or some high order structures, can be in different tautomeric forms and chemical degradeted by light effect, temperature change and in time. PROBLEMS

  11. DNA's (bp) Molar extinction coefficients  (max)‏ stDNA 12680 M-1cm-1 (260 nm)‏ Tyr Tyrosine 1,440 M-1cm-1 (274 nm)‏ Phe Phenylalanine    220 M-1cm-1 (257 nm)‏ PROBLEMS (Continuation) The analysis of the obtaining concentration dependences in the future needs the known values for concentrations of both A and B components very accurately determined.

  12. Some curious mistakes can be sometimes in your protocol of an experiment You want to research the dimarization of a drug in solution Next example You are preparing a series of solutions containing a drug at different concentrations Kd A2 2A You record, for example, fluorescence emission spectra of these solutions And we want to obtain good result by optimization of spevtra and ... + Kd

  13. Some errors in our concentration dependencies can be through non calibrated instruments Relative C/Cint The calibration curve is not a linear one 0.5 • For calibration, we prepare a series of solutions containing the analyte in the concentration range expected • Record the fluorescence emission (FL) of each solution • Construct plot the data (relative C/Cint vs. FL) • Compare obtained curve with line 0.4 0.3 0.2 0.2 0.1 FL intensity 0 200 250 300 50 150 100

  14. Thus, good concentration dependencies we can obtain by titration procedure. In main biophysics methods this method of titration is used more offen. Case 2 CB0 is fixed V1 ofCA0 is added to V2 of CB0 At this method of titration, concentrations of both A and B components are differed from mixture to mixture and observed results are interpreted not so lightly CA0 CB0

  15. Hard to understand why treatment of numerous experimental data obtained from biophysical methods to determine thermodynamic parametrs of complex formation are done up to now with the use of the model of one type of complex. Of cource, there are different kinds of bound drugs to DNA (NA), but we can assume that in a drug-NA mixture several types of complexes are formed n=6-8 bp n<1 bp n=2-4 bp

  16. model of one type of complex up to now describes the all biophysical experimental data

  17. Stern – Volmer equation Io/I = 1 - KC Io/I-1 CL0 = 1 model of one type of complex fluorescence 0 C

  18. McGhee equation 1/Tm - 1/T0 = R/m1 - KC) 1/n 1.0 model of one type of complex model of one type of complex 0.5 T Tm T0 – DNA alone Melting of DNA in the prsence of a drug

  19. G= -RT lg K G= -TS DSC, T is canged ITC, T is fixed model of one type of complex model of one type of complex model of one type of complex Differential scanning calorimetry Isothermal titration calorimetry

  20. We compare usually our results with results detected in another methods CD polarimetry DSC, ITC calorimetry model of one type of complex for a system so on and values of binding parameters estimated should be the same ones fluorometry spectrophotometry

  21. By titration we obtain the set of spectral data that should be large enough to be statistically treatment by new algorithms (for example, either by method of chemometric analysis or by rather like methods) actinocin – ctДНК , CA0=2x10-5 M What is P/D? P/D is the molar ratio of DNA base to drug Kruglova E.B., Gladkovskaya N.A., and Maleev V.Ya. Use of Spectrophotometric Analysis to Calculate the Thermodynamic Parameters of Binding between an Actinocin Derivative and DNA. Biofizika 2005. V.50. 253

  22. Beer-Lambert Law A = ( l ) cwhere:A  - absorbance at wavelength  - Molar absorptivity at , M-1 cm-1c - concentration, M Principle of additivity: Absorbance of mixture at 1 should be the sum of the absorbances of the components at 1 A(mixture) 1 = A(1) 1 + A(2) 1

  23. Block-scheme of our algorithm  1 19 CA,10, CB,10 block of calculation of equilibrium concnetrations A1,1 A1,n K, n CA,20, CB,20 A2,2 A2,n . . . . . . . . . . . . . . . . . K,  CA,n0, CB,n0 An,1 An,n block calculation of absorptions of mixtures, Aij block of comparisonof Aij exp. and Aij cal. exit Criteria of conformity of a model to experimental data k [Ck ]i

  24. A+ Bn ABn K1 2A A2 Kd 2( ABn ) ( ABn )2 K2  1 1 19 19 A+ Bn ABn CA,10, CB,10 A1,1 A1,n K CA,20, CB,20 A2,2 A2,n . . . . . . . . . . . . . . An,1 An,n CA,n0, CB,n0 McGhee, von Hippel Model

  25. Lipfert J., Klijnhout S., and Dekker N.H.Torsional sensing of small-molecule binding using magnetic tweezers. Nucleic Acids Res. 2010 November; 38(20): 7122–7132. Force-induced stretching DNA elongetion in the presence of ethidium bromide (EtBr): concnetration dependences Temperature induced melting of DNA

  26. So, at concentration of EtBr CetBr< CEtBrcrt the stretching curves exhibit cooperative transition effect similar to that seen in the absence of the drug. r/CEtBr But we have no peculiarities in this range of Scatchard isotherm. Why? At CetBr< CEtBrcrt the force extension curves are simple, monotonically increasing functions. This CetBrcrtto be CetBrcrt = 2,5*10-8 M r

  27. Vladescu I.D., McCauley M.J., Rouzina I. And Williams M.C. Mapping the phase diagram of single Dna molecule force-induced melting in the presence of ethidium. 2005. Phys Rev. Lett. V.95. . It would be very interesting to find the answer to the question what is CetBrcrt = 2,5*10-8 M ???

  28. These questions wait to our answers

  29. Thank you

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