UV Fluorescence of Photosensitive Porphyrins – Liberation from Chlorophylls by Alkaline Lysis

Photosynthesis is one of the most beautiful thing which surrounds us in our everyday life. I wish other people could also see the green leafs and mosses by the way I do. Understand, or try to understand, what happens under the surface. Not only the fine and elegant structures of photosystems, and the biochemistry of chloroplasts, but the way how quanta of light energy seem to move through many possible routes simultaneously in photosystems is mesmerizing. The quantum way of scanning multiple chlorophyll molecules simultaneously by light energy triggered excitons can explain the great efficiency of life, unlike the classical way of seeing things hopping from one place to another. The intuitive way of seeing world seems to be in contradiction to reality. So, because of pure curiosity and a couple interesting ideas on my mind, I wanted to try to liberate the magnesium chelating porphyrins from chlorophylls and chloroplasts while preserving their photosensitivity.

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First, I smashed some green leafs of fern with a spoon on a piece of plastic wrap. Then I put the homogenate in glass tubes.

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In one tube I added pure water as a control. Another tube was filled with saturated sodium bicarbonate solution, and the third one was filled with acetic acid solution. The tubes where shaken vigorously and the green turbidity were observed in all the tubes.

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Then I used my fancy centrifuge for having clear supernatants, free of plant debris.

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Green turbidity is still present. I should have check the intensities of Tyndall effects in the samples for comparison. The scattering of light might result from intact cell organelles for example. More centrifugation would also be good of course, and to use some additives like detergents and maybe some precipitating agents.

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Finally, it is time to turn the lights off, illuminate the samples with UV LED and see the results.

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Here is the water treated control sample. The purple color has slightly shifted to green, to longer wavelength, meaning lower energy, but not much. It seems that without additives, like acids, bases, or detergents, the chloroplasts and/or photosystems in lipid rafts might still be somewhat functional, and the absorbed light energy is used for metabolism not totally impaired yet. However, if I did not imagine, the intensity of the green fluorescence increased a little during time. That is maybe due to proceeding necrosis, why the metabolism loses its strength to consume supplied light energy and/or the liberation of porphyrins and their higher structures continues.

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Here is the acetic acid treated sample which did not fluorescence at all. There is a clear reason for this. The acidity might destroy the metabolism of the chloroplasts, it can liberate the porphyrins to more soluble form by detaching the hydrophobic phytol tails, but the chelated magnesium ions are also replaced by protons, making the phorphyrins photodeactive. That is why only purple Tyndall effect can be seen here, and no chromatic shifts.

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Tadaa! Here is the sodium bicarbonate treated sample. First, see the intense green fluorescence, coming from the soluble photosensitizes (likely the magnesium chelating porphyrins). Second, see how surface localized the fluorescence is. According to Beer-Lambert law, there is a hell of an amount of soluble porphyrins (or chlorophyll containing lipid structures) in the solution, as the light cannot penetrate deep into the solution. The base has lysed the plant tissues a little, and destroyed the light energy consuming metabolic machinery. Even the antioxidant system, like glutathiones, which should scavenge the radicals formed by the photosensitizes should now be oxidized. So, the chemical route is somewhat stuck, why the porphyrins are in an excited state for a while after receiving photons, but then some of the energy is lost in thermal vibrations and the leftover energy is re-emitted away; the chromatic shift from purple to green can be seen. I hope that this is not due to the presence of carbon dioxide, formed by the reactions between organic acids and the used bicarbonate. For being sure about this, next, I should try some other base instead of sodium bicarbonate. A stronger base would help the lysis also. If the reason for the result is only in the alkaline lysis of plant tissues, meaning the better liberation of phorphyrins’ higher structures, a control sample with appropriate detergent could be tried, which could solubilize porphyrins in a form or another (as chlorophylls in micelles and lipid rafts) into the supernatant. However, the cool thing about the alkaline mechanism in the molecular level is that like with the acids, bases should also detach the lipophilic phytol tails from the porphyrins, making them more soluble instead of being shackled into the highly amphipathic structures (chlorophylls), but by preserving the chelated magnesium simultaneously.

 

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