Quarterly Technical Report April 1, 1952 Thru June 30, 1952

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TEE UNIVERSITY OF ROCHESTER Atomic Energy Project Rochester 20, New York QUARTERLY TECHNICAL REPORT 1%should be noted t h a t the Quarterly Technical Reports of The Universi do not attempt t o describe progress i n i n those i n which some significant resul ich are not sufficiently complete t o be written up Date of Report: 7/31/52

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CONCEBNIHG TBE APPLICATION OF ION EXCHANGE RESINS M STUDIES OF COMPLEX IONS The method for determining the dissociation constants o f complex ions by use of cation exchange resins and radioactive isotopes has been elaborated to include cases where the complex contains more than one metal atom and also where the metal i o n polymerizes in solution. The use of cation-exchange resins and radioactive isotopes for the determination o f dissociation constants of metal-containing complex ions has received much attention (1). Thus far, the equations on which the ion-exchange method of studying complex ions is based-presuppose that the complex ions under study are mononuclear and that the free metal ions are unpolymerized (2). The application (3) of this technique t o a system which at times, depending on pH, contains polynuclear complex ions and polymerized metal ions has made it necessary for us to formulate a more generalized equation than has previously appeared. Consider the dissociation of the complex i o n The cation M may be polymerized to the extent of 2, and the anion is assumed to be in a simple form. The thermodynamic dissociation constant may be written as: where parentheses, brackets and Y represent activity, concentration and activity

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If the Y ratio remains constant we obtain the Kc: If charge -c i s zero or negative and M goes on the resin in the same form i n which it exists in solution we obtain the follow i ag : When no complexer A is present: where -v is t h e volume of solution, 5 is the mass of resin used, and $ is the number of moles. I n the presence of reactant A:

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A study of equation (8a) will show that only i n the case, x = a does the term drop out. Equation (8a) then reduces t o the equation of Schnbert e t a l . and the absolute ion concentration o f M need not be determined. In such a caae, y may be determined by aplot of l / K a vs. [AT i n which the proper value of y is used t o give a straight line. 6ome power of the ligand concentration and a straight l i n e is obtained, this is cannot be determined unless the value o f a i s determined by some independent means. This is i n contrast to the method of continuous variations (4, 5 ) by which the ratio y/x between the anion and cation in the complex may be determined even when a i s unknown. However, the ratio y/x between M and A in the complex If the metal ion polymer is broken up as it goes onto the r e s i n according t o the following reaction: the final equation obtained is aleo (sa).

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1. Schubert, J., J. Pbys. Chem., 56, 114 (1952). This paper has the complete Energy Project Report No. UR-198 (1952). Job, P., Ann. Chim., (10) 2. 113 (1928), and A m . Chim., (11) 6, 97 (1936).

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COPCERNING THE! PRESEHCE OF CITRATE IN COMMERCIAL CRYSTALLIZED BOVIm SERUM ALBUMIN Isaac Feldman and Jean R. Havill Each of two batches of A n n o u r ~ sCrystallized Bovine Serum Albumin investigated contained a significantly large amount of strong anionic complexing agent. It is to the extent believed that this contaminant is of at least 0.5 moles per mole of in any theoretical study i n which become a widespread custom to use citrate and that it is present albumin. This substance ahould the commercial preparation is Armourrs Crystallized Bovine Serum Albumin, without pretreatment, as a representative protein in the etudy of the theory of ion-protein binding. blood by the procedure of Cohn e t al. (1). that all but an insignificant amount of the citrate is removed by the cryetallization lium by albumin, it has been discovered that the commercial albumin preparation contains a significantly large amount of a strong complexing agent. Considering the relative binding strengths of the various possible contaminants, it is believed that this anion is citrate and is present to the extent of at least 0 . 5 moles per mole of protein. This preparation is obtained from citratedIt appears to be generally ,aeaumed However, during the course of an investigation of the binding of berylBeryllium begins to hydrolyze a d polymerize near pH 4.6 even in concentrations as low as (See Table I, Page 10) the concentration of beryllium in barbiturate buffer at pH 7.5 and 6Oc. which is capable of diffusing through a cellophane bag ifJ -M (2). Under the conditions of the present experiments

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chloride (spiked with radioactive Be7 isotope) to 296 Armour's Crystallized Bovine Serum Albumin in buffer were dialyzed versus 10-ml. volumes of buffer, the concentration of diffusiblesberyllium increased to about 1 - 2 x 10 It was possible to decrease the amount of beryllium-solubilizer associated I with the protein to a very small, but still detectable, value by dialyzing SO ml. of 8% cryfitalline albumin in a rotating cellophane bag versua 20 litere of buffer (0.025 -M barbiturate, 0.125 NaCl) two successive times at 6 O c . for 24 hours each time. As is seen in lines 6 and 7 of Table I, Page 10, 1.3 x -M I beryllium i s diffusible in the presence of this predialyzed preparation. calculation from the data of lines 2, 7 and 8 of Table I, Page 10, contaminant was still preaent even after the dia1rSi8 treatment. show8 that roughly 4% of the original The seriousness of the presence of such a strong complexlng agent must be considered in any theoretical study in which the commercial preparation is need, even if the identity of the contaminant is not posit'ively known at present. The following results, however, suggest that the contaminant is probably citrate. From a consideration of the crystallization process of the albumin, it 6eems that the only beryllium-solubilizing agents which c d d possibly be preeent i n appreciable concentration are citrate, acetate, bicarbonate, and proteindecompo6ftion products such as amfno acids. It is evident from the data of Table If, Page 11, that, of these substances, only citrate could conceivably be in sufficient concentration to account f o r the diffusibility of beryllium in t h e Dresence of the un-predialyzed crystallized albumin. Since near pH 7.5 one citrate ion can bind a maximum of two beryllium atoms ( 3 ) , one can calculate the probable minimum amount of citrate present in

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the crystalline protein. albumin contains at least enough citrate to bind three micromoles of beryllium; i.e., at least 1.5 micromoles of citrate. be eqected, a citrate/albumin ratio greater than 0.5 seems to occur. The data of Table I indicates that 2.8 micromoles of Because some mass-action effect is t o It is also of interest that a sample of Fraction V albumin contained a considerable amount of beryllium-solubilizing agent even after dialysis of 50 m l . of a 25% solution in a rotating cellophane bag three successive times versus 20 liters of distilled water at 6OC. each time. equal volume of buffer, all the beryllium remained in a diffusible state.. The significance of these results with respect to beryllium-protein binding of this albumin and 6 x beryllium was dialyzed versus an will be discussed elsewhere,

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Diff usi bil it y of Beryllium i n Presence of Commercial Crystalline Bovine Sem Albumin, pH 7.5, 6OC. 10 m l solution prepared i n buffer (.O5M barbiturate 0.10 M NaC1) dialyzed versus 10 m l . buffer for 16 hours on BGerner Oscillating Flatform Shaker If 100% d if fusible * Be concentration determined by radioactive isotope counting technique (2). ** Bo. 1 and Eo. 2 represent two different batches of Amour's Crystalline Bovine Serum Albumin.

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Diffusibility of Beryllium in Presence of Solubilizing Agents Possibly Present in Commercial Crystalline Bovine Serum Albumin, pH 7.5; 6OC. beryllium plus added possible complexers dialyzed versus 10 ml of buff% for 16 hours on Boerner Oscillating Platform Shaker 1 Concentrations expressed in moles/liter x lo4 *IAdded Substance, Total. Conc. Sodium Acetate 100. and 1000. (dl and 1 aspartic acid and ) Be Outside of bag at eqail.** * Initial concentration in bag + 2. ** Be determined by radioactive isotope counting technique (2). *** Parentheses indicate number of erperiment6 averaged.

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TBE ILOFLUENCE OF POLYPHOSPHA~SON URANIUM EXCRETION The urinary excretion of uranium was enhanced by the cyclic polyphosphate polymers while the other polymers either depressed the excretion or had no affect at all on this metal. The salt employed either the c&cium or sodium. . salt and the route of administration a l s o had marked affects. These polyphosphates influenced the urinary excretion of uranium only over the first twenty-four hours. Fecal excretion of uraniupl was not appreciablr enhanced in the single observation that was made. Purpose: It has been found by in vltro studies that the uranyl ion _ _-- forms P, relatively stable complex ion with a variety of phosphate polymers rats and rabbits (2, 3) and are relatively non-toxic (4, 3, 5 ) . These polyphosphates have also been found to be rapidly excreted by-- :-. basis further studies were made to determine if certain phosphates might possess antidotal properties in experimental uranium poisoning. Their affect on the toxicity of uranium was investigated (4) concurrently w i t h a study of their influence on the excretion of the uranyl ion. This report summarizes o n l y the latter studies which represent short-term observations after single intraperitoneal doses of uranium in the rat. Chemistry: The uranyl nitrate, made up as a one molar stock solution, was diluted in order that a volume of about one m l , given to rats by the intraperitoneal route, contained a sublethal amount of uranium (1mg U/kg) . Several cyclic and straight chain phosphate polymers were tested;

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Sodium Tetrwtaphosphate - Victor Chemical Works (E-5707) Sodium Trimetaphosphate - Victor Chemical Works (E-2664) Sodium Victamide - Victor Chemical Works (0-4502) same source as NO. 6) Sodium Tripolyphosphate - Victor Chemical Works (0-4502) Eimer and Amend (S-307) All of these salts were injected either subcutaneously or intraperitoneally i n sub-lethal doses of 100 mg P/kg or. lees. sodium salt was dissolved i n two t o five milliliters of water and adjusted t o a pH of 7.4 using either dilute H C l or FISOH. Sodium orthophosphate represented a mixture of NaH2po4 and NazHpo4 a t a pH of 7.4. Each dose of The soluble Ca-I?a HMP w a s prepared from solutions of the sodium salt by the addition of CaC12, t o yield a ' final Ca/P mol ratio of 0.30. Ca BMP, Calcium phytate and Calcium Victamide were administered as white precipitates suspended i n water; their Ca/P mol ratios were 0.50 Procedures: I n the excretion studies, only addt male albino rats Generally the injection of the uranyl nitrate and the polyphosphateswere used. were given simultaneously; although, i n some 'cases, the latter were dao given daily for a period of two or three days. or two r a t s were placed i n each glass metabolism cage for twenty-four hours. During this time they received water ad libitum but generally no food. The feces and urine were separated. The cages were then washed dam with

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dilute Hm? and the washings added t o .the urine s aq les. were evaporated t o a small volume and vet ashings were accomplished with were diluted t o volumes appropriate f o r analysis (generally 100 ml). When free of organic material, the solutionsm occasions, the feces were also ashed with a mixture of HNO3, H2SO4 and H202. Analysis: Two methods f o r analyzing ashed urines were used t o determine the quantity of uranium excreted. required the enrichment of the uranyl nitrate solution with $35 before A plating and counting technique (6) which injection and the fluorophotanetric method (7) i n which no enrichment w a s necessary. The latter proved more satisfactory. When determinations are made by the fluorophotometric method, such inorganic elements as calcium and perhaps phosphorus are known (7) t o quench in part the fluorescence developed by uranium. Table I records the magnitude of this effect in normal rat urine and i n urine supplemented with excessive amounts of calcium and phosphorus. These added amounts of calcium and phosphorus (series B anc C ) were greater than the doses of phosphates employed in this study. Thus the analyses for uranium were not significantly biased by the additional amount of these two elements i n samples from phosphateResults: Figure I demonstrates that rats receiving certain polyphosphates and U02(U03)2, excreted more u r a n i u m than did rats which were given administration of the cyclic polymers (sodium trimetaphosphate and sodium This enhanced excretion was noted only after the i.p. tetrametaphosphate) . The linear polymers (HMP and tripolyphosphate) and phtate and Vietamide either did not influence the amount of uranium excreted during the first day or reduced it below that i n the control group. A reduction w a s obtained whenever both polyphosphates and Uo;Z(UO3)2 were given

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Table I: Uranium recoveries by fluorophotomtric analysis on ashed urine containing excessive amounts of calcium and phosphorus. Uranium Uranium Phosphorus calcium Recovery Average * Each sample represents the t o t a l daily urine of two male rats (300 gms. each). These samples were supplemented with ham amounts of uranyl nitrate and CaH4(po4)2, as indicated. Before these additions, each sample contained Table 11: Daily Fecal Excretion of Uranium (uranium given i .p .) Per Cent of Dose Found in Feces

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by the same route (intraperitoneal); even orthophosphate was effective in of administration (the latter subcutaneous), the excretion of uranium was camparable with that in the control group. When uranium and polyphosphate were given by different routes Sodium and calcium salts were not compared by the subcutaneous route of administration since the sodium salt of HMP (and presumably that of the other polymers) was poorly tolerated subcutaneously (2). When these salts were given intraperitoneally, however, the sodium polymers were probably more effective than the calcium salts in depressing the excretion of uranium. terms of uranium excretion however, the route of administration of the line= polymers was probably more important than the associated cation. As indicated in Figure 11, the polyphosphates were able to affect the excretion of uranium only during the first twenty-four hour period after administration of this metal. Thereafter, the amounts found in the urine were comparable in treated and untreated groups. Fecal excretion of uranium, which is always s m a l l …