A simple method, based on ethanol fractionation, for the preparation of highly
purified human serum albumin with a higher yield than that of the conventional ethanol
procedures is described. It consists of two purification steps, namely, precipitation of most
of the other plasma proteins from a 3-fold diluted plasma with ethanol at 42% concentration,
pH 5.75 and -5°C, leaving over 96% pure albumin in the supernatant, followed by isolectric
precipitation of albumin from the supernatant at pH 4.8 and -5°C. The paste thus obtained
was processed to the final albumin solution according to the conventional methods. The yield
of the final albumin with a purity of over 99% was equivalent to 29.5 g/l of plasma representing
a recovery of over 93%. The possibility of recovering other plasma proteins and the suitability
for large scale preparation are also discussed.
The effect of oleate, diphenylhydantoin, and heparin on the binding of 125 I-thyroxine to highly purified human thyroxine-binding globulin (TBG) has been studied. Oleate was about twice as effective as diphenylhydantoin in displacing 125 I-thyroxine from TBG as ascertained by starch gel electrophoresis at pH 8.6. In vitro addition of heparin was relatively ineffective in inhibiting thyroxine (T4) binding to TBG. The possible physiological significance of the results lies in the observation that oleate inhibited T4-binding to TBG at molar ratios to T4 that were well within the normal range (free fatty acid: T4) existing in plasma. (/ Clin Endocrinol Metab 36: 392, 1973) HPHE concentration of free fatty acids (FFA) •*-in human plasma is known to fluctuate widely in response to a variety of dietary and hormonal stimuli. For example, the plasma level of FFA is increased by fasting (1), in uncontrolled diabetes (2), and after administration of epinephrine (1,3), norepinephrine (4), growth hormone (5), and thyroid hormone (6). It has also been shown by Hollander et al. (7) that induction of high FFA levels in human plasma both in vivo and in vitro leads to a rise in the concentration of free thyroxine (T4). The authors (7) attributed the rise in percent free T4 to competition between FFA and T4 for thyroxine-binding sites in plasma. They also suggested that fluctuations in circulating levels of free fatty acids could serve as a regulator of peripheral thyroid hormone action by influencing the binding of thyroxine in plasma (7). Since thyroxine-binding globulin (TBG) is the major T4-binding protein in plasma (8), it was of interest to investigate the effect of a representative fatty acid such as oleate on the interaction of T4 with purified TBG. In addition the effects of diphenylhydantoin and heparin on the binding of T4 to TBG have also been studied.The highly purified human TBG used in these experiments is the same TBG preparation previously described (9,10). As noted previously (9), the binding capacity of the TBG was less than 1 mole T4 per mole TBG; the maximal T4-binding capacity approached a limiting value of 0.25-0.3 mole T4 per mole TBG.Starch hydrolyzed for gel electrophoresis was a product of Connaught Research Laboratories, Toronto, Canada and was purchased from Fisher Scientific Co. 125
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