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George Hogeboom and Walter Schneider


Experimenting with phenolphthalein
Christian de Duve Scientist
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And so we injected this dye to the rabbits. Now, it turns out that the liver in most animals, including ours, will convert many foreign substances that are injected in the bloodstream... will convert them... or even some normal substances to the glucuronides which is... which are compounds in which glucuronic acid, which is derived from glucose but is an acid, of course, is attached to the compound by glycosidic bond, so it's a glucuronyl... a better glucuronyl compound X. And these glucuronides are then excreted in the urine. That's the way we detoxicate a number of substances: to solurilise them we combine them to glucuronic acid and then the compound is eliminated in the urine. And so these rabbits would detoxicate the phenolphthalein with glucuronide, and... glucuronic acid, and they would excrete in their urines phenolphthalein glucuronide, the compound. I still see those... you know, urine has a tendency to become alkaline because of the production of ammonia, and so red... red... so their cages were covered with red urine. Anyway, we collected the urine and from the urine isolated the glucuronide and the glucuronide was used as substrate to assay the enzymes. Again, it was quite a job and that's very easy. Once you have the glucuronide, all you have to do is to... to incubate with the enzyme; then you add some alkaline, and then you measure the red colour, because a free phenolphthalein reacts with alkaline – becomes red – but the glucuronide remains colourless, so it is very easy. Nowadays this is done not with phenolphthalein but with methyl-umbelliferone derivative which can be assayed by its ultra violet absorption. Anyway, that's... Gianetto did that, and we compared acid phosphatase with beta glucuronidase, and indeed it behaved, essentially, in the same way. Not exactly... there was a problem... there was more enzyme in the microsomal fraction, but that's a different story. But basically what came down with the mitochondria would come out together with acid phosphatase... it was latent; would come out together with acid phosphatase. When we released one, we released the other.

Belgian biochemist Christian de Duve (1917-2013) was best known for his work on understanding and categorising subcellular organelles. He won the Nobel Prize in Physiology or Medicine in 1974 for his joint discovery of lysosomes, the subcellular organelles that digest macromolecules and deal with ingested bacteria.

Listeners: Peter Newmark

Peter Newmark has recently retired as Editorial Director of BioMed Central Ltd, the Open Access journal publisher. He obtained a D. Phil. from Oxford University and was originally a research biochemist at St Bartholomew's Hospital Medical School in London, but left research to become Biology Editor and then Deputy Editor of the journal Nature. He then became Managing Director of Current Biology Ltd, where he started a series of Current Opinion journals, and was founding Editor of the journal Current Biology. Subsequently he was Editorial Director for Elsevier Science London, before joining BioMed Central Ltd.

Tags: Robert Gianetto

Duration: 3 minutes, 8 seconds

Date story recorded: September 2005

Date story went live: 24 January 2008