Monitoring Ligand Binding with Ultrasonic spectroscopy

Binding of Mg2+ to polyriboadenilic acid, poly (A)

Binding of cytidine 2'-monophosphate (2'-CMP) to ribonuclease A (RNase A)

 

 

Binding of Mg2+ to polyriboadenilic acid, poly (A)

A concentrated solution of MgCl2 was added stepwise into the measuring ultrasonic cell containing 1 ml of aqueous solution of poly(A) (analogue of well known RNA) and into the reference ultrasonic cell containing 1 ml of buffer. The device measured the difference in ultrasonic parameters of the samples in the measuring and the reference cells, thus subtracting the contribution of MgCl2. Therefore the plotted changes of ultrasonic velocity and attenuation represent the interaction of magnesium ions with poly(A) only.  Binding of magnesium with poly(A) results in the initial decrease of ultrasonic velocity caused by the release of hydration water from the coordination shell of Mg2+ ions and atomic groups of the polymer. The compressibility of water in the hydration shells of the ligand and the polymer is less than the compressibility of the bulk water, therefore transferring of hydration water into the bulk water increases the total compressibility of the solution, thus reducing the ultrasonic velocity. When all available sites on the polymer are occupied by the ligand the curve levels off. The total drop in ultrasonic velocity is linked with the number of water molecules excluded from the coordination shell of Mg2+ allowing to make structural characterisation of the complex. Binding constants and stoicheometries can be calculated from the shape of the curve. At high concentrations of magnesium electrostatically neutralised polymer molecules begin to form aggregates. The scattering of ultrasonic waves by the aggregates leads to the increase in attenuation. Additional dehydration and intrinsic compressibility of the aggregates results in the decrease of ultrasonic velocity at this stage. It is important that all these measurements do not require any optical activity of the ligand and the polymer and optical transparency of the medium.  As the hydration, or solvation, effects are involved in most of reactions in solutions the high-resolution ultrasonic spectroscopy has a potential of being a universal technique for their analysis.

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Binding of cytidine 2'-monophosphate (2'-CMP) to ribonuclease A (RNase A)

2'-CMP inhibits RNase A activity, being isomeric with the natural RNA structure. The binding site for 2'-CMP lies deep within the cleft of the enzyme.

A concentrated solution of 2’CMP was added stepwise into the measuring ultrasonic cell containing 1 ml of aqueous solution of protein and into the reference ultrasonic cell containing 1 ml of buffer. The device (HR-US 101) measured the difference in ultrasonic parameters of the samples in the measuring and the reference cells, thus subtracting the contribution of 2’CMP. Therefore the plotted changes of ultrasonic velocity represent the interaction of the ligand with protein only.  Binding of 2'-CMP to enzyme results in the initial decrease of ultrasonic velocity caused by the release of hydration water from the coordination shell of ligand and atomic groups of the polymer. The compressibility of water in the hydration shells of the ligand and the polymer is less than the compressibility of bulk water, therefore transferring of hydration water into the bulk water increases the total compressibility of the solution, thus reducing the ultrasonic velocity. When all available sites on the polymer are occupied by the ligand the curve levels off.

The total drop in ultrasonic velocity is linked with the number of water molecules excluded from the coordination shell within binding site allowing to make structural characterisation of the complex. Binding constants and stoichiometry can be calculated from the shape of the curve.

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