A biuret method for determination of protein in hyamine hydroxide and NCS solutions used for liquid scintillation counting in toluene
✍ Scribed by M. Schmukler; M.J. Yiengst
- Publisher
- Elsevier Science
- Year
- 1968
- Tongue
- English
- Weight
- 236 KB
- Volume
- 25
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
An aqueous alkaline protein solution is not miscible with toluene. Therefore, in order to measure the radioactivity of protein samples by liquid scintillation counting in a toluene solution, two nonaqueous solvents have been developed: Hyamine hydroxide (14) and NCF (5). Although Hyamine hydroxide is itself miscible with water, when a Hyamine-protein solution is added to water, a heavy precipitate results. The addition of NCS alone to water produces a heavy precipitate. Because of this, protein determination in Hyamine hydroxide and NCS solutions cannot be accomplished with presently available assays, all of which are performed in an aqueous medium. This paper describes a nonaqueous biuret reaction that permits protein measurement in organic solvent systems, thus allowing radioactivity and protein determinations to be made on aliquots of the same sample.
Methods
Bovine serum albumin and purified rat liver protein were used as protein standards in these experiments. BSA2 was obtained from Sigma Chemical Company. Rat liver protein was prepared as follows: Liver was homogenized in 5% TCA. The pellet was twice extracted for 15 min at 70°C in 5% PCA. It was then washed with 95% ethanol and extracted 5 min at 50" in l/l ethanol/chloroform.
The pellet was then washed with l/l acetone/ether and ether, and then vacuum-dried.
Hydroxide of Hyamine-10X was obtained from Packard Instrument CO. and NCS from Nuclear-Chicago Corporation.