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. 2000 Jun 20;97(13):7325–7330. doi: 10.1073/pnas.97.13.7325

Figure 4.

Figure 4

HPLC elution profiles and activity of solutions containing iron, hypoxanthine, and uracil. Preparations of hypoxanthine/uracil/Fe2+ complexes were subjected to HPLC. Each preparation contains peaks corresponding to the active initial void-volume fraction in natural stomatin preparations (arrows) and peaks comigrating with free hypoxanthine and uracil. Activity of test solutions is expressed as % activity of stomatin (3 mg/ml). (A) Elution profile of solution (50 μl) containing hypoxanthine, uracil, and FeSO4 prepared based on the procedure described by Albert (24). The mobile phase was H2O at a flow rate of 0.5 ml/min. (B) Activity of the initial void-volume fraction indicated by the arrow in A (0.17 A250 units/μl) compared with the activity of a fraction prepared in identical manner but using a solution from which uracil was omitted (H-Fe, 5 μl, 0.13 A250 units/μl) and a fraction from which hypoxanthine was omitted (U-Fe, 5 μl, 0.26 A250 units/μl). The same results were obtained when 10 μl were added. Hypoxanthine (50 μM), uracil (50 μM), FeSO4 (100 μM), and H2O were substituted for stomatin as controls. (C) Elution profile of solution prepared by a procedure modified from Nelsen et al. (17). The injection volume was 50 μl and the mobile phase was H2O at a flow rate of 0.5 ml/min. (D) Activity of different concentrations of the unfractionated iron-base solution in C compared with the activity of hypoxanthine (50 μM), uracil (50 μM), and FeSO4 (100 μM), tested separately (H, U, Fe) and in combination (H + U + Fe). The activity of solutions made by this method varied, but differentiation as high as 96% of the stomatin control has been obtained. (E) Elution profile of solution containing 2 μg of the complex prepared by modification of the procedure described by Acevedo-Chávez et al. (25). The mobile phase was 50 mM citrate buffer at a flow rate of 0.5 ml/min. (F) Activity of different final concentrations of the unfractionated complex prepared by the method of Acevedo-Chávez et al. (25) and dissolved in H2O and adjusted to pH 5 with KOH.