The interactions of Ca2+ ions with plasma proteins were first studied by Pribram (1871) Mg2+, Ca2+ > Sr2+> Ba2+ ions, although Mg2+ ion is often irregular in position (Schubert, 1954).In this paper an attempt has been made to extend this work by comparing the binding of the alkaline-earth ions to native and chemically modified human albumin over a range of pH and in the presence of near-physiological concentrations of Na+ ions. Additional information about the nature of the binding sites on albumin for these ions is given. Greenwald (1926) Measurement of radioactivity. The method of isotope dilution was used for the quantitative determination of metal ions. The isotope 28Mg was counted with a wellcrystal system in conjunction with an automatic scaler.The isotopes 45Ca and 89Sr were counted by a p-particle liquid-scintillation technique at -17°. Diphenyloxazole (0-3 %, w/v) in toluene was used as the organic scintillator. Aqueous samples (1 ml.) were blended with a scintillator (5 ml.) with ethanol (10 ml.). Protein solutions were precipitated by the ethanol in the counting solution. When the precipitated protein was allowed to settle in the counting dish it did not appreciably affect the count. In practice, therefore, the protein was precipitated and kept for at least 3-4 hr. in the dish at -17°before counting. Solutions containing buffer ions in the presence and absence of protein were counted with the same efficiency as an aqueous solution containing the same amount of activity. Each sample was counted for a minimum of 150 000 counts.Equilibrium dialysis. All dialyses were carried out with 10 ml. of 0-5-0-7% (w/v) protein-buffer solution inside Visking cellulose membranes. The outer solution (120-140 ml.) was of measured pH and molarity 0-15-0-17.Each dialysis was done in a dialysis rocker overnight at room temperature. Control experiments showed that with this apparatus 0-10m-sodium chloride came into equilibrium with water in 3 hr. Dialysis experiments as a function of pH were carried out at fixeed Ca2+, Mg2+ and Sr2+ ion concentrations of 20, 10 and 40 mg./l. respectively.Outer buffer solutions. The following buffer systems were used: pH 2-4, 0-05M-potassium hydrogen phthalate; pH4-6, 0-05M-sodium acetate-acetic acid; pH6-7-5,0-02M-sodium cacodylate; pH 8-9-5, 0-lm-sodium hydroxideboric acid-potassium chloride. Sodium chloride was added to each buffer solution to bring the molarity to 0-15-0-17.Protein concentration. Protein was estimated by a semimicro-Kjeldahl technique; a protein:N factor of 6-25 was assumed.

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