Abstract
A few years ago one of the authors in unpublished results showed that rhodamine-B would precipitate pepsin from solution. Since the well known proteolytic ferment precipitating agent, safranine, also precipitates insulin, 1 it was decided to determine if rhodamine-B would precipitate insulin and if so, whether the dye could be removed without inactivation of the insulin. Our experimental results show that insulin is readily precipitated by rhodamine-B at pH 7.2. The complex is soluble in distinctly acid solution, probably with decomposition into its components, for the dye can be practically completely removed by extraction with isoamyl alcohol, leaving the insulin behind in the aqueous layer.
To 1 cc of insulin, Lilly, (40 units per cc) 1 cc of a phosphate buffer solution of pH 7.2 was added, followed by 1 cc of a saturated solution of technical rhodamine-B which was either centrifuged or allowed to stand for several days to remove water insoluble impurities. Almost immediately after addition of the dye a flocculent precipitate separated out. After standing for approximately 10 minutes the precipitate was removed by centrifuging and washed with approximately 3 cc of the buffer solution. The washed precipitate was then dissolved in 1 cc of 0.1N HC1 and diluted to 4 cc. Typical blood sugar changes following its subcutaneous injection into rabbits are shown in Table I. Although the number of animals used was not sufficient to give accurate data on the percentage recovery they do show, however, that it is reasonably satisfactory.
In other experiments, after the insulin-rhodamine-B complex was dissolved, the solution was extracted several times with isoamyl alcohol to remove the rhodamine-B. These extractions left a faint pink color in the aqueous layer which we were unable to remove by additional extractions.
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