The interplay of proteins and peptides with inorganic material is a fundamental phenomenon with implications for nanotechnology, biomaterials and biotechnology. The first step in comprehending of such phenomenon is revealing the fundamental physico-chemical constants such as adsorption constant, Gibbs free energy, enthalpy, entropy, and limited adsorption which may be evaluated through establishing adsorption isotherms. However, adsorption from liquid phase is restricted with kinetics, surface capacity, pH, and comparative adsorption which all should be consciously considered before setting the experiment.
In this video, my colleagues Elena Korina and Sergei Neifert will present the physico-chemical study of dipeptide adsorption on solution disperse titanium dioxide covering preparation features that will help avoid hidden risks which researchers may face while performing relevant experiments. Place 183 milligrams of dipeptide into the sterile polymeric test tube and dilute up to approximately 35 milliliters with double distilled water and dissolve at room temperature under vigorous stirring. If the dipeptide doesn't dissolve in double distilled water and stirring, place the dipeptide solution into the ultrasonic bath and sonicate for a few minutes.
Adjust the pH of pre-dissolved solution of a peptide to 7.4 by cautiously adding solution of MES or sodium hydroxide to the dipeptide solution upon stirring at room temperature and monitoring the pH with a pH meter. After adjusting the pH, pour the solution into measuring cylinder. Rinse the test tube and fill the measuring cylinder with the double distilled water up to 40 milliliters to make the final concentration of 16 millimolar.
Prepare dipeptide dilutions with concentrations between 0.4 and 12 millimolar by diluting 16 millimolar dipeptide solution with double distilled water. For example, in order to prepare eight millimolar dipeptide solution, add seven milliliters of double distilled water to 10 milliliter of 16 millimolar dipeptide solution. After dilution, adjust the pH to 7.4 by adding drop-wise solution of MES or sodium hydroxide to the dipeptide solution.
After adjusting the pH, pour the solution into the measuring cylinder. Rinse the test tube and fill the measuring cylinder up to 20 milliliters with double distilled water to make a concentration of eight millimolar. Other dilutions of dipeptide stock solution are prepared accordingly.
In the end, we get a row of dipeptide dilutions ready for adsorption studies. Prepare 10 millimolar MES buffer solution. Adjust the pH to 7.4 with trisodium hydroxide upon stirring and monitoring the pH with a pH meter.
This solution will be used for the sole preparation. Grind approximately 200 milligrams of nanocrystalline titanium oxide in a mortar for at least five minutes. Weigh 40 milligrams of grind titanium dioxide nanoparticles.
Put the flask into the sonication bath using the laboratory stand. Add the grind titanium dioxide in 20 milliliters of MES buffer into the flask using the glass funnel and sonicate in an ultrasonic bath for 20 minutes. Set the thermostat to the desired temperature.
Add one milliliter of the sonicated sole of titanium dioxide to the marked adsorption vials. Place the marked adsorption vials against corresponding dilutions in an improvised floating device made of styrofoam and put it into the thermostat to equilibrate the temperature for at least five minutes. After that, add one milliliter of dipeptide dilution to the corresponding marked adsorption vial making sure all mixing solutions have same temperature.
Keep the series of obtained adsorption samples to the thermostat for 24 hours to achieve the adsorption equilibrium. Occasionally agitate titanium oxide dispersions during thermostating. In order to avoid temperature-induced re-equilibrium, take out one sample from the thermostat for filtration at a time.
Take a sample of the dipeptide solution from each glass vial with a syringe. Remove the needle from the syringe and put on the syringe filter to filter the dipeptide solution into the glass vial. Repeat the filtration with samples of other concentrations.
Now these samples are ready for analysis. Make the 50 milliliter solution of TFA in acetonitrile. Spike 0.34 milliliters of TFA in the measuring cylinder and adjust the volume of solution to 50 milliliters with acetonitrile at room temperature.
Prepare the derivatization solution. Spike 299 microliters of phenylisothiocyanate and 347 microliters of triethylamine in the measuring cylinder and fill the cylinder up to 50 milliliters with acetonitrile. Prior to the high performance liquid chromatography analysis, derivatize the samples with Edman's reagents in the chromatography vials.
Mix the 400 microliters of the sample with 400 microliters of derivatization reagent. Keep the samples at 60 degrees for 15 minutes. After heating, neutralize the samples with 225 microliters of TFA solution and wait for a few minutes to cool sample to room temperature.
Use HPLC analysis to determine the concentration of the dipeptide solution before and after the adsorption. Start the analysis of the samples with the necessary conditions which are set by the software. The dependencies of adsorption from the equilibrium dipeptide concentration after adsorption, adsorption isotherms were plotted accordingly to the obtained experimental data.
The measurements of dipeptide adsorption were data processed using the Henry model. The equilibrium binding constant was obtained from the slope of the dependence of dipeptide adsorption on the dipeptide equilibrium concentration. The van't Hoff equation was used for determining the standard Gibbs free energy for each temperature.
Plot in a graph of free energy versus temperature, we determined the enthalpy as interception of the linear graph with a free energy axis for dipeptide. The change in entropy for each temperature was determined from the fundamental equation. The calculated values of equilibrium binding constant, standard Gibbs free energy, enthalpy and entropy for dipeptide are presented in table one.
Adsorption isotherm construction from depletion data remains to be the most available methodology that does not require expensive setups, providing exhaustive physico-chemical data for literally every soluble sorbate. Combined with spectroscopic or computer-based data, it may reveal fundamental structural features of complex behavior of biomolecules upon contacting the inorganic nanoparticles.
