1. Alkaline-earth-metal cations at low concentrations form soluble complexes with bovine caseins. The relative order of binding capacities is: Mg(2+)>Ca(2+)>Ba(2+)>Sr(2+). 2. The cations interact with both free ionized carboxyl groups of aspartic acid and glutamic acid and with monoester phosphate groups covalently bound to serine and threonine; at low concentrations of the cations interactions are predominantly with the phosphate groups. 3. The order of binding capacities for purified components of the casein complex is: alpha(s1)-casein>beta-casein>kappa-casein.

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.

1.Studies on the binding of scandium to human apotransferrin showed that two Sc3+ were 2. The binding of each Sc3f involves the ionisation of two phenolic tyrosyl residues. 3. The scandium * protein complex has similar resistance to denaturants and chemical reagents 4. The only protein complex observed, when quantities ofSc3f below the free iron binding capa-5. The injection of Sc3+, as a 1:2 citrate complex, into rabbits led after 48 h to all the plasma bound specifically a t the two iron-binding sites.as the iron complex. city of serum were added to whole serum in vitro, was the Sc3+ * transferrin complex.Sc3+ being present as the transferrin complex.Previous studies on the metabolism of scandium were initiated through possible hazards arising from its presence in nuclear explosion sites [l], because of its chemical similarity to yttrium and the lanthanides, whose biological properties have been extensively The &globulin, transferrin, in its pure form was shown to bind a number of trivalent metal ions, including scandium [8]. Transferrin was also implicated in the transport of various metals in the plasma, apart from iron, including chromium MATERIALS AND METHODS Materials MethodsDifference spectroscopy readings were taken on a UnicamSPSOO recording spectrophotometerfittedwith a scale expansion unit. All other spectrophotometric readings were taken with a Gilford 2000 spectrophotometer fitted with a Unicam SP500 monochromator. Radioactivity measurements of solutions containing 46Sc or S9Fe were made using a y scintillation spectrometer. All protein concentrations were determined using an E~~~ = 9.23 x lo4 11131 (the on binding Sc3f to tranferrin is less than lo/, as shown below). For binding measurements by gel filtration, apotransferrin (5 ml, 50/, w/v) in 0.1 M Tris-C1 buffer a t the appropriate pH was equilibrated, for either 1 h a t 23 "C or one week a t 4 "C, with a ten-fold excess of Sc3f as a 1 : 2 Sc3f -citrate complex containing 10 pCi of 4 %~. Excess metal was removed on a 35 ~2 cm column of Sephadex G-25 equilibrated with 0.1 M Tris-C1 buffer. Fractions (3 ml) were collected and the protein eluates localised and metal concentrations determined by radioactivity measurements.The scandium -transferrin samples thus obtained (from the longer incubation period only) were passed down a 2 0 x 2 cm column of Chelex 100. Fractions were treated as for the Sephadex 6-25 column.Ultraviolet difference spectra upon metal binding were recorded using dual compartment cuvettes. I n one compartment was placed protein (5O/, w/v) in pH 8.0 0.2 M Tris-C1 buffer; in the other 2.0 mM scandium and 4.0 mM trisodium citrate in the same buffer. For the titration of scandium and apotrans-

In a previous structural study of stability constants of oc-amino acid metal complexes (Perkins, 1952) the work was restricted to amino acids closely related to glycine. In this paper the work is extended to a wide range of a-amino acids to discover the effect of different functional groups. Most of the amino acids are closely related to a-alanine and norvaline. Albert (1952) has published stability constants for the complexes of a number of these amino acids with a selection of metal ions. In the present work only the metals beryllium, zinc, cadmium and mercury of group II of the periodic classifieation were used. EXPERIMENTAL Materials. The following A.R. salts were used in 0-01M solution: BeSOj, 4H20; ZnSO4, 7H20; 3CdSO4, 8H,20. Hg(NO3)2 was made up in 0-025a-EHNO, and excess acid back-titrated in the presence ofthe amino acid investigated.The amino acids were obtained from British Drug Houses Ltd. and Roche Products Ltd., except for 8-aminovaleric acid, for a sample of which I am indebted to Dr T. S. G. Jones and Dr S. Wilkinson of the Welcome Research Laboratories, Beckenham, Kent. All the amino acids were dried in vacuo at 100°for at least 48 hr., with the exception of cysteine hydrochloride, asparagine and glutamine which were dried in a vacuum over silica gel for 48 hr. The purity of each amino acid was checked by paper chromatography.Titrations. The ionization constants of each amino acid were determined by the potentiometric method of Albert (1950) in 001 M aqueous solution, except for tyrosine and diiodotyrosine which were determined in 0{002m solution.Titrations in the presence of metallic ions were usually carried out in 001 M amino acid and 0-005 M metal solution. The exceptions were tyrosine and diiodotyrosine, which were used in 0-002ms solution, and cysteine and histidine, which were used in 0-0025 s solution.Calculations. The two systems to be considered in caloulating the stability of each complex are:(a) M2+ + amino acid = (complex I)+, with dissociation constant = K1.