History
By 1923 Theodor Svedberg and his student H. Rinde had successfully analyzed large grained sols terms of their gravitational sedimentation. Sols consist of a substance evenly distributed in another substance, also known as a colloid. However, smaller grained sols, such as those containing gold, could not be analyzed. To investigate this problem Svedberg developed an analytical centrifuge, equipped with a photographic absorption system, which would exert a much greater centrifugal effect. In addition, he developed the theory necessary to measure molecular weight. During this time, Svedberg’s attention shifted from gold to proteins.
By 1900, it was generally accepted that proteins were composed of amino acids; however, whether proteins were colloids or macromolecules was still under debate. One protein being investigated at the time was Hemoglobin. It was determined to have 712 carbon, 1,130 hydrogen, 243 oxygen, two sulfur atoms, and at least one iron atom. This gave hemoglobin a resulting weight of approximately 16,000 Da but it was uncertain whether this value was a multiple of one or four (dependent upon the number of iron atoms present). In regards to Svedberg’s studies, hemoglobin was the main protein of interest.
Through a series of experiments, utilizing the sedimentation equilibrium technique, two important observations were made: hemoglobin has a molecular weight of 68,000 Da, suggesting that there are four iron atoms present rather than one, and that no matter where the hemoglobin was isolated from, it had exactly the same molecular weight. How something of such a large molecular mass could be consistently found, regardless of where it was sampled from in the body, was unprecedented and favored the idea the proteins are macromolecules rather than colloids. In order to investigate this phenomenon, a centrifuge with even higher speeds was needed, and thus the ultracentrifuge was created to apply the theory of sedimentation-diffusion. The same molecular mass was determined, and the presence of a spreading boundary suggested that it was a single compact particle. Further application of centrifugation showed that under different conditions the large homogenous particles could be broken down into discrete subunits. The development of centrifugation was without a doubt an important turning point in protein science.
Read more about this topic: Centrifugation
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