How did they know what we knew? like in the case of TCA cycle

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AllenChiu's picture
How did they know what we knew? like in the case of TCA cycle

There is a question that has bothered me for a long time,which is "how did they know what we knew?" in the early time researches of metabolism,like the understanding of TCA cycle in1940s when there was barely any DNA/protein information or investigation technology.I mean no NCBI,no WB,no molecular biology.
So how did they know what they knew?What kinds of experienments did they do?Is there any way that I can see any of their original(raw) reports(publications) at that time?
Thank you very much for any clues.

protoldo's picture
Hola, I have lived the

Hola, I have lived the transition of metabolic studies to molecular biology. The main difference is that before the studies were done with mamalian enzymes (proteins) but not in humans. For instance, if we wanted isolate the citrate syntetase, we started of high volume of rat, pig, rabbit or cow liver and a colorimetric method for detection of citrate was developed to mesure the enzimatic activity. The extract was sequentially precipitated with ammonium sulphate and an active fraction  was isolated, for instance 40-60% amm. sulphate. afterwards a isoelectric pptation was made f.i. ph 7.2. Other phisical process like extraction in organic solvents, centrifugation in density gradients were used. These steps were increasing the purity and specific activity of enzyme, but decreasing volume of sample. At the 70s ion exchangers and gel filtration resines and PAGE  were used and they  were an important help to get pure proteins. When molecular biology  come, these studies were moved to humans and with the immobilitazion of antibodies and tags designs we get our days. I don´t know specific papers of these methods but for instance Ochoa S. studied a lot of enzymes. I suppose that famous names as Krebs Watburg or any names of metabolic routes developped biochem assays to get the conclusions that gave their fame.Have a nice day