Phosphorylation assay in liquid scintillation counter using C̆erenkov radiation of 32P: Application to photophosphorylation
✍ Scribed by Kozi Asada; Masa-aki Takahashi; Mieko Urano
- Publisher
- Elsevier Science
- Year
- 1972
- Tongue
- English
- Weight
- 284 KB
- Volume
- 48
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Application to Photophosphorylation
Gerenkov radiation produced by hard p-emitters has been applied to the assay of radioisotopes in a liquid scintillation counter without the addition of scintillators (1,2). In aqueous samples, the P-rays having energies greater than 0.263 MeV are expected to produce Gerenkov radiation (3). In the case of xzP, most of the ,&rays do this. The assay of 3?P in a liquid scintillation counter by i'erenkov radiation (4-8) has an advantage over conventional methods in that a large counting volume is permitted while retaining a relatively high counting efficiency, which is not affected by pH or the presence of salts. In the present communication, an assay that employs this advantage is described for determining photophosphorylation. The procedure is applicable to assays of other types of phosphorylation.
Materials. Any "'P-containing impurities in the 3zP-orthophosphate were removed as follows: The "'P-phosphate supplied commercially in 0.1 M HCl was autoclaved at 1 kg/cm' overpressure for 1 hr after addition of 10 pmoles NaH,PO,. The solution was diluted with water to reduce the HCl concentration below 10 mM and applied to a small column (0.4 X 2 cm) of Dowex 1 (Cl-). The column was flushed thoroughly with water, followed by 10 ml 20 mM HCl. The "Pi was then eluted with 10 ml 50 mM HCl.
Spinach chloroplasts were prepared and stored at -20" in 507% glycerol as described elsewhere (9).
Procedure for Photophosphorylation Assay. The basic react'ion mixture contained 50 mM Tricine-KOH buffer, pH 8.0 (25"), 10 mM NaCl, 5 mM 32Pi (about lo6 cpm) , 1 mM ADP, 4 mM MgCl,, and chloroplasts containing 30 ,ug chlorophyll in a total volume of 2 ml unless otherwise specified. In addition the mixture contained 0.01 mM phenazine methosulfate (P&IS system), 0.1 mM NADP, and 8.5 ,uM spinach ferredoxin 311 @ 1972 hy Ac:~dclnic Prcrs. 11~.