The overall goal of the synthesis and purification is to relatively easily produce a ruthenium 360 analog used in mitochondrial calcium uptake assays that is 10 times more effective than commercially available ruthenium 360. This method can help answer key questions in the field of mitochondrial calcium biology by providing a pure inhibitor of mitochondrial calcium uptake. The main advantage of this technique is that the final product is both very potent and pure.
Individuals new to this method may struggle because purification requires time and patience. Demonstrating the producer will be Sarah Nathan, a graduate student from my laboratory. Begin this procedure with synthesis of pentaammine chloro ruthenium dichloride as described in the text protocol.
Dissolve 100 milligrams of pentaammine chloro ruthenium dichloride in 50 milliliters of one molar ammonium hydroxide solution in a 200 milliliter round bottom flask. Loosely cap the flask with a glass stopper and heat the reaction mixture at 75 degrees Celsius for six hours. Heating a sealed vessel results in a pressure build up.
Make sure the glass stopper is loose to allow release of excess pressure. After six hours remove the reaction mixture from the heat. And stir at room temperature for four days to yield a dark green solution.
To purify the resulting trans-diaqua octo amine mu-oxo diruthenium pentachloride by cation exchange chromatography first suspend five grams of mesh cation exchange resin in 10 milliliters of 0.1 molar HCL in a 25 milliliter beaker. Load this slurry into a 10 milliliter column affixed with a 50 milliliter solvent reservoir. Wash the resin with approximately 20 to 30 milliliters of 0.1 molar HCL until the elute is colorless.
Next add concentrated HCL to the previously isolated green reaction solution to adjust the PH to two at which point the solution color changes to brown. Load this acidified solution to the cation exchange resin column by gently pipetting it on top of the resin. Let the elute completely drain and continue loading the solution.
Repeat this process until the entire solution has been added. The top of the resin will be dark brown or black. The resin will decrease in volume slightly.
Next cover the top of the resin with glass beads to prevent the resin from being disturbed when new solutions are added. Elute the column with 20 milliliters of one molar HCL. Then elute the column with 50 milliliters of an increased HCL concentration of 1.5 molar.
A yellow solution will begin to come off the column. Next increase the HCL concentration to two molar and continue eluting with 150 to 200 milliliters. The eluant will start as a very pale yellow and go to a bright yellow.
Wait until the eluant coming from the column is again very pale. Patience is critical at this step. The eluant must be very pale.
Increase the HCL concentration further to 2.5 molar. Collect the eluant as fractions in test tubes. Finally increase the concentration to three molar HCL.
The product will elute from the column as a green-brown solution. A red-brown fraction may also begin to come off the column. These fractions are ruthenium brown impurities and should not be pooled with the green-brown fractions.
To test all of the fractions by UV-Vis spectroscopy add 100 microliters of a given fraction into two milliliters of three molar ammonia. Then analyze the mixture by UV-Vis spectroscopy. Fractions containing pure product will have a large absorbance band at 360 nanometers and a less intense absorbance at 600 nanometers.
Absorbance at 480 nanometers is indicative of ruthenium brown and absorbance at 533 nanometers represents ruthenium red impurities. Other impurities are seen at absorbance of less than 300 nanometers. Pool fractions containing pure product and evaporate the solution to dryness by rotary evaporation before analysis of the sample in the solid state by IR spectroscopy.
Proceed to evaluate mitochondrial calcium uptake inhibition by fluorescent spectroscopy as detailed in the text protocol. UV-Vis spectra of purified trans-diaqua octo amine mu-oxo diruthenium pentachloride is characterized by the major and minor absorptions at 360 nanometers and 600 nanometers respectively. Conversely an impure mixture shows absorption bands at 533 nanometers from ruthenium red impurities and at 275 to 300 nanometers from starting material.
Solid state characterization using infrared spectroscopy has the expected stretches from the amines. As well as the ruthenium oxygen ruthenium stretch at 850 inverse centimeters. Permeabalized HeLa cells treated with a calcium sensitive fluorescent dye and trans-diaqua octo amine mu-oxo diruthenium pentachloride show inhibition of calcium uptake into the mitochondria.
While attempting the procedure it is important to remember to allow the fractions to completely elute. Patience will yield a pure product. After watching this video you should have a good understanding of how to synthesize, purify and characterize this potent ruthenium 360 analog.
Mitochondrial calcium levels regulate a wide range of intercellular processes including bioenergetics and controlled cell death. The inhibitor produced from this method can be applied in studies to understand the role of mitochondrial calcium biology in mediating other biological processes. Don't forget that working with concentrated acids and bases are corrosive and precautions such as wearing proper PPE should always be taken while performing this procedure.
