Evidence for the Reduction of Histone as the Mechanism of Action of Toroglobulin
Two methods describing the Histone Reductase Reaction by Toroglobulin (Bull. Am. Phys. Soc., Mar.2017 Meeting, B4, New Orleans) (2), are illustrated. The first is electron spin resonance, which supports the paramagnetic interaction of a spin field between Toroglobulin and Histone 2S. The second is Cyclic Voltammetry, showing the direction and amount of the charge transfer.
Electron spin resonance (ESR) was performed on a Resonance Model 8400 X-Band Spectrometer. All samples are frozen overnight in NMR tubes. Toroglobulin is prepared as a stock 2.0 % aqueous solution. Histone 2-S is from Sigma, and prepared as a 5.0 mg/mL aqueous solution. NaCl is from Sigma and is prepared as .9 % aqueous solution.
The reaction tube is a mixture of .2 mL Toroglobulin, .2 mL Histone 2 S, and .4 mL NaCl. Control NMR tubes are established for Toroglobulin with NaCl, and Histone 2S with NaCl. On removal from the freezer (19 degrees F.), the tube is inserted and measured in the spectrometer. The ESR scans are printed and reviewed on finely calibrated drafting paper. The reaction is reviewed with the insights of V.B. Fedorov on paramagnetic relaxation, in : Quantum Electronics and Paramagnetic Resonance, D.K.Skobel’tsyn, Ed. (3), Proceedings of the Lebedev Physics Institute, V.49, 1971. In magnetically concentrated systems the paramagnetic relaxation mechanism is complex. There are two limiting pathways of energy transfer. In the first the spin excitation is transformed into the energy of thermal motion by the spin-lattice interaction of the Zeeman degrees of freedom with the lattice. In the second case the energy is transferred first from the Zeeman system to the spin-spin degrees of freedom, and then to the lattice. Only the combined Toroglobulin, with Histone 2S, in saline, produced positive ESR data. This ESR scan produced a symmetric quartet of spikes as the hyperfine splitting (fig. 4). The distance between lines is 10.4 Gauss, for a total of 40.16 Gauss. These data show that Histone 2S and Toroglobulin are electron spin resonant and therefore capable of charge exchange.
Next we measure the direction and the amount of the charge transfer between Toroglobulin and Histone 2S. For this, Cyclic Voltammetry of the reaction is performed with an EG&G Parstat Potentiostat. We use a gold working electrode, a Pt counter electrode, and a Ag/KCl reference electrode. The background electrolyte is .1M NaAcetate. Nitrogen purging is ten minutes. A 416 mv. peak shift of Toroglobulin (from +68 mv to +484 mv) as a response to the charge addition (-) by Histone IIS, is shown on the horizontal axis (fig.5). The Toroglobulin current as read on the vertical axis is seen to be diminished. We see that the histone is acting as an electron acceptor. Toroglobulin, the electron donor, is acting as a histone reductase. If we divide the 416. mv. peak shift by the Nernst electron transfer equivalent of 59. mv, there are approximately seven electron transfer equivalents in this reaction.
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