We studied similar samples using EPR spectroscopy. Tell me more about your measurements.... We have excellent Bruker EPR. I will try to help you.

Dear colleague,

I use to study protein-bound (ubi)semiquinones by EPR spectroscopy. If you would like to know the parameters I use, you could refer to my papers (e.g. Grimaldi S et al. , Biochemistry 2001) or you could send me an email directly ([email protected]) to get more details. Please don't forget to tell me what kind of EPR spectrometer you have. Regarding the preparation of such samples, it seems that Dr. Pavel Stopka is by far more experienced than me.

Best regards

Dr. Stephane Grimaldi

Thank you Dr. Stopka and Dr. Grimaldi for your answers and your help. We have a Bruker EMXmini 6/1 spectrometer with a 6" magnet, PremiumX microwave bridge, standard ER4119HS resonator, and the Xenon acquisition software. We are currently using about 20 to 60mg muscle biopsy samples frozen in liquid nitrogen. In a finger dewar we are sandwiching the sample between two cylindrical pieces of frozen 0.9% saline solution (about 15mm long). A cotton swab is inserted to hold the sample in place and liquid nitrogen poured on top to keep everything frozen. This is inserted to center the sample in the cavity. The settings are: C.F. 3358 G, sweep width 200 G, sweep time 12 seconds, receiver gain 39 dB, mod. amplitude 4 G, M.W. power 10mW, conv. time 24 ms, and time constant 81.92ms. Any suggestions would be appreciated. Dr. Grimaldi, I will look up your article. Thanks.

Dear Dr. Reese,

some years ago we detected ubisemiqquinones in isolated rat heart mitochondria (RHM). Concentrated suspensions of RHM in the presence of susbtrates were rapidly frozen in cylindrical quartz tubes (4 mm OD) by liquid nitrogen and then measured at 200K

Typical parameters we used were: microwave frequency 9.43 GHz, power 2 to 20 mW, Mod frequency 100 kHz, mod amplitude 4 G, receiver gain 4 * 10^5, points 1024, time constant 0.655 s, scans 4, sweep 80 G, center field 3360 G.

Further details you will find in : H. Nohl, L. Gille, K. Schonheit, and Y. Liu. Conditions allowing redox-cycling ubisemiquinone in mitochondria to establish a direct redox couple with molecular oxygen. Free Radic.Biol.Med. 20 (2):207-213, 1996.

Although I wish you much success with your experiments I have some doubts whether ubisemiquinone concentrations in muscle biopsies are high enough to be detected. Even if you detect signals it remains the questions whether you really detect ubisemiquinones. In mitochondria a running respiration (substrate) prior to freezing is required to detect semiquinone intermediates. So I guess for muscle biopsies also the mode of freezing ist important.

So far I know only about the EPR detection of ubisemiquinones in isolated mitochondria, submitochondrial particles and isolated enzyme complexes.

Best regards

Lars Gille

As you probably found, there are remarkably few publications on ubisemiquinone radicals in tissues at physiological pH. It sounds easy to do - EPR spectroscopy is very sensitive to radical signals, and there are lots of species in cells (quinones, flavins, phenols, ascorbate, etc.) which in principle can produce them.

For simple low-mass quinone compounds in solution, the radicals can sometimes can be generated at very alkaline pH. Their EPR spectra can be measured at room temperature and low power; they are very pretty spectra with multiple hyperfine lines.

But this doesn't work with ubiquinones in biological systems. For one thing, they are membrane-bound, and effectively immobile on the EPR timescale, so the spectra are broadened out.

Another important reason is that, at equilibrium, the semiquinone state is less favoured thermodynamically, relative to the quinone and quinol states. So the semiquinone signal is there, but at undetectably small concentration.

The exceptions are proteins where the semiquinone is stabilized by the enzyme mechanism, The most successful studies have been on isolated complexes prepared under the right redox conditions, for example succinate-ubiquinone reductase (see for example PMID: 1309986). In this protein the radical signal shows rapid relaxation owing to an interaction with an iron-sulfur cluster, hence cryogenic temperatures are needed.

It also needs some work to demonstrate that the signal is not due to any of the other radical-producing compounds.

Perhaps some background and more information would be helpful. Our lab is interested in the free-radicals produced during exercise, in various disease states (COPD, chronic heart failure), and in normal aging; with and without anti-oxidant supplementation. Our biopsy samples are taken, placed in a tube and flash frozen in liquid nitrogen. Because these biopsy samples are used later for other analyses such as fiber typing and mitochondrial assays, and because we are interested in the oxidative state of the tissue when each sample was taken, they are kept frozen during preparation and EPR analysis. Our scans are done at about 100K. As mentioned, there is very little research published on ubisemiquinones in intact tissue. Davies et al reported a signal at around g=2.004 in tissue homogenates that they later identified as two semistable forms of ubisemiquinone. I believe a few others have also reported a similar result. We have also seen a peak at g= 2.004, but it is barely above background and is not a clean signal. Since we are interested in the free-radical profile in situ (if you will allow me to use the term here) with intact tissue, it might be difficult to resolve these issues.

We have been able to clearly observe an organic radical signal using EPR spectroscopy in both intact cells and isolated mitochondria. When we simulated the EPR spectra, we were able to quantify the radical signal using SpinCount and it was found to be < 1uM. However, we used temperatures in the range of 4-10 K, established by use of liquid helium. At higher temperatures, the signals get much weaker and the radical is not observable.