J. H . Stern and L. R. Beeninga
and kindness in reading the manuscript prior to publication and for his helpful suggestions.
References and Notes (1) A. L. Goodliffe, H. D. B. Jenkins, S. V. Martin, and T. C. Waddington, Mol. Phys., 21, 76 (1971). (2) H. D. B. Jenkins and T. C. Waddington, J. Chem. Phys., 58, 5323 (1972). (3) H. D. B. Jenkins and T. C. Waddington, Nature (London),Phys. Sci.. 232, 5 (1971). (4) H. P. Dixon, H. D. B. Jenkins, and T. C. Waddington, J. Chem. Phys., 57, 4388 (1972). ( 5 ) H. P. Dixon, H. D. B. Jenkins, and T. C. Waddington, Chern. Phys. Lett., I O , 600 (1971). (6) H. D. B. Jenkins and T. C. Waddington, J. lnorg. Nucl. Chem., 34, 2465 (1972). (7) A. Neckel and G. Vinek, Z. Naturforsch. A, 28, 569 (1971). (8) W. Van Gooi, J. Bruinink, and P. H. Bottelberghs, J. lnorg. Nucl. Chem., 34, 363 1 (1972). (9) M. F. C. Ladd, Trans. Faraday Soc., 85, 2712 (1969). (IO) H. D. B. Jenkins and T. C. Waddington, Nature(London),Phys. Sci., 238, 126 (1972). (11) M. F. C. Ladd, Nature(London), Phys. Sci., 238, 125 (1972). (12) M. Mori, R. Tsuchiya, E. Kyuno, and T. Nichide, Bull. Chem. SOC.Jap., 33, 1510(1960). (13) A. B. Blake and F. A. Cotton, lnorg. Chem., 2, 906 (1963). (14) J. Beck, R. H. Wood, and N. N. Greenwood, horg. Chem., 9, 86 (1970). (15) E. F. Bertaut, J. Phys. Radium, 13, 499 (1952). (16) H. D. B. Jenkins, Chem. Phys. Lett., 9,473 (1971). (17) C. E. Moore, Nat. Bur. Stand., Circ. No. 467 (1958). (18) H. Von Halben and M. Litrnanowitsch, Helv. Chim. Acta, 24, 44 (1941). (19) A. VisteandH. B.Gray, horg. Chem.,3, 1113(1964). (20) W. H. Waggoner and M. E. Chambers, Talanta,5, 121 (1960). (21) J. A. Campbell, Spectrochim.Acta, 21, 1333 (1965). (22) L. Oieari, G. De Micheiis, and L. Di Sipio, Mol. Phys., 10, 111 (1966).
(23) J. E. Mayer, J. Chem. Phys., 1, 270 (1938). (24) J. R. Tessman, A. H. Kahn, and W. Shockiey, Phys. Rev., 92, 890 (1953). (25) M. L. Huggins, J. Chem. Phys., 5, 143 (1937). (26) L. Pauiing, Z.Kristallogr.,8, 377 (1928). (27) R. W. G. Wyckoff, "Crystal Structures," Voi. 3, 2nd ed, interscience, New York, N.Y., 1965. (28) A. Niggli, Acta Crystallogr.,7, 776 (1954). (29) K. Herrnann, M. Hosenfeld, and N. Schonfeid, Wlss. Veroff. Siemenskonz, 5, 119 (1926). (30) M. Y. Colby, Z.Kristallogr., 78, 168 (1931). (31) W. H. Zachariasen and G. E. Zeigier, Z. Kristallogr.,80, 164 (1931). (32) H. W. Smith, Jr., and M. Y. Coiby, Z. Kristallogr., 101, 90 (1940). (33) J. J. Miller, 2.Kristallogr.,99A, 32 (1938). (34) C. N. Muidrow, Jr., and L. G. Hepier, J. Amer. Chem. SOC.,79, 4045 (1957). (35) F. D. Rossini, eta/., Net. Bur. Stand., Circ., No. 500 (1952). (36) S.S. Batsanov, Zhur. Neorg. Khim., 9, 1322 (1964). (37) G. De Michelis, L. Oieari, and L. Di Sipio, Coord. Chem. Rev., 1, 18 (1966). (38) I. H. Hilller and V. R. Saunders, J. Chem. SOC.0,1275 (1969). (39) I. H. Hiiiier and V. R. Saunders, Proc. Roy. Soc., Ser. A, 320, 161 (1970). (40) D. D. Wagrnan, W. H. Evans, V. B. Parker, i. Haiow, S.M. Bailey, and R. H. Schurnrn, Nat. Bur. Stand., Tech. Note, No. 270-4 (1969). Jap., 12, 241 (1957). (41) M. L. Huggins and Y. Sakamoto, J. Phys. SOC. (42) D. F. C. Morris, Proc. Roy SOC.,Ser. A, 242, 116 (1957). (43) T. C. Waddington, Advan. horg. Chem. Radiochem., 1, 157 (1959). (44) G. V. Sarnsonov, Vysokotemp, 110 (1965). (45) K. B. Yatsimirskii, *OX, 28, 9 (1956). (46) M. F. C. Ladd and W. H. Lee, J. lnorg. Nuci. Chem., 21, 216 (1961). (47) K. B. Yatsimirskii, J. Gen. Chem. USSR, 28, 2655 (1956). (48) K. B.Yatslrnirskii, Zhur. Obshch. Khim., 28, 2376 (1956). (49) N. M. Selivanova and M. Ch. Karapet'yants, lzv. Vyssh. Ucheb. Zaved., Khlm. Khim. Tekhnol., 8, 891 (1963). (50) M. F. C. Ladd and W. H. Lee, J. horg. Nucl. Chem., 30, 330 (1968). (51) H. D. 8.Jenkins, J. Chem. Phys.,J8, 5969 (1972).
Partial Molal Heat Capacities of Caffeine and Theophylline in Pure Water J. H. Stern* and L. R. Beeninga Department of Chemistry, California State University, Long Beach, California 90840 (Received Ju/y 3 1, 1974; Revised Manuscript Received December 9, 1974)
Calorimetric enthalpies of solution of the biochemically important methylated xanthines, caffeine and theophylline, to very low concentrations in water have been measured from 288 to 308'K. The temperature derivatives of the enthalpies yield the appropriate partial molal heat capacity differences between the aqueous solutes and the pure crystalline solids, with values of 100 and 90 cal/deg mol a t 298'K for caffeine and theophylline, respectively. These results, combined with the estimated heat capacities of the crystalline solids, give the anomalously high partial molal heat capacities of 160 and 140 cal/deg mol at 298'K foi caffeine and theophylline, respectively, and are evidence of the complex structuring induced by the two solutes. The CH2 group contribution to the partial molal heat capacities deduced from these two xanthines is approximately the same as that for various aqueous aliphatic nonelectrolytes, showing that limited group contribution predictions for partial molal heat capacities of aqueous nonelectrolytes may be possible when more data becomes available.
Introduction Aqueous methylated xanthines are important biochemical solutes1 with very interesting but poorly understood physicochemical properties.2 A previous paper3 included a study of the enthalpies of solution of crystalline caffeine in pure water a t 298'K, as a function of concentration. This contribution reports on the partial molal heat capacities of caffeine and theophylline at very low concentrations in The Journai of Physical Chemistry, Vo/. 79, No. 6, 1975
pure water via measurement of the enthalpies of solution from 288 to 308'K. The interaction of these solutes with water and their effect on aqueous solution structure should be of particular interest since both xanthines have similar pharmacodynamical properties, particularly as diuretic^.^ 'It has been observed that the anomalous partial molal heat capacity difference ACO,, between a pore and a dissolved solute at low concentrations in water, or the absolute partial molal heat capacity of the aqueous solute Gop2,
Heat Capacities of Caffeineand Theophylline
583
TABLE I: Enthalpies of Solution of Caffeine and Theophyllinein Pure Water
I
No. of runs
288 298" 303 308
11 22 6 11
I
CHJ
caffeine 1,3,7-trimethylxanthine
T , OK
A H o z (obsd), AHoz (calcd),
kcal/mol
kcal/mol
Caffeine
CHJ
theophylline 1,3,dimethylxanthine
are very sensitive indicators of aqueous solution structure changes. The few AC",, values for aliphatic nonelectrolytes which have been reported to date5-1° are positive, and generally increase with the size of the nonpolar groups or number of alkyl groups in the nonelectrolyte. The positive value of ACO,, can be interpreted as evidence of increased structure induced in the water by the presence of the dissolved nonelectrolyte. The systems of aqueous nonelectrolytes upon which these conclusions are based include a few alkanes, hydrogen bonding hydrophilic low molecular weight aliphatic alcohols, alkyl a ~ i n e s ethyl , ~ ~ ~a ~ e t a t e ,nitro~ methane,6 and acetic acid.7 This study appears to be the first involving the effect of two related heterocyclic ring compounds on water.
288 293 298 303 308 Reference 3.
10 2 5 3 7
3.00 i 0.06
3.60 i 0.06 4.20 i 0.12 4.97 i 0.14 Theophylline 3.91 i 0.07 4.12 i 0.18 4.67 i 0.01 4.94 0.03 5.80 i 0.19
*
3 .OO 3.61 4.19 4.97 3.91 4.15 4.54 5.07 5.76
amination of heat capacity data for low molecular weight aliphatic alcohols and amines,s>g alkyl ammonium, and fatty acid saltslo shows that AC",, increases by approximately 15 cal/deg mol per CH2 group added. It appears then that the CH2 group contribution is approximately constant, whether in an aliphatic or heterogeneous aromatExperimental Section ic ring compound. In this connection it may be noted that The calorimeter and calorimetric procedure have been measurements with the biochemically similar isomer of described elsewhere.ll The pure crystalline anhydrous caftheophylline, theobromine (3,7-dimethylxanthine), were feine was identical with that used in the previous study3 unsuccessful because of the very slow rate of dissolution in and the anhydrous theophylline was obtained from the Nuwater. However, in view of the observed regularity, it can tritional Biochemical Corp. Elemental analysis of theobe assumed that the heat capacity behavior and consephylline gave the following percentage results: calcd, C, quently the effect on aqueous solution structure of the two 46.85; H, 4.48; N, 31.10; 0, 17.76: found, C, 46.85; H, 4.58; isomers may be the same or very similar. N, 30.94; 0, 17.51. In each run 480 g of distilled and deionIt would be desirable to obtain absolute values of the ized water were weighed into the dewar to f0.05 g. partial molar heat capacities Cop, of the aqueous solutes by combining AC",, with the heat capacity of the crystalline Results and Discussion C,,(s). However, direct exsolids Cop2(s):Cop, = AC",, The means of the observed enthalpies of solution of cafperimental values of C,,(s) are not available for caffeine feine and theophylline in pure water, AH"2 (obsd), with and theophylline. Estimated values of C,,(s) at 298"K were their standard deviations are shown in Table I. The very (xanthine) calculated as follows: C,,(s) (caffeine) = C,,(s) low overall final molality in this study ranges from ca. 0.002 3C,,(s) (CH2) and C,,(s) (theophylline) = C,,(s) (xanto 0.006 m. No variation in enthalpies over this concentrathine) 2C,,(s) (CHz), where C,,(s) (CH2) is the CH2 contion range was observed within the'limits of the reported tribution to the heat capacity of the solid. The experimenuncertainties. The enthalpies may thus be considered as tal value of Cpn(s)(xanthine) is 36.5 cal/deg m01.l~The calinfinite dilution values, with no evidence of possible culated value of C,,(s) (CH2) was obtained from the differself-association that may take place at higher concentraence between the experimental values of the heat capacities The calculated enthalpies, A H o 2 (calcd), in of crystalline 2-methylnaphthalene and naphthalene:16 kcal/mol are from eq 1 and 2 for caffeine and theophylline, C,,(s) (CH2) = C,(s) (2-methylnaphthalene) - Cp2(s) (naphthalene) = 47.2 - 39.7 = 7.5 cal/deg mol. In this way AH",(caffeine) = 314.6 - 2.185T 3.83 x 10-3~2 the estimated values of CP&) were calculated to be 59 and (1) 52 cal/deg mol for caffeine and theophylline, respectively. AH",(theophylline) = 238.9 - 1.667T + 2.95 x It may be noted that Kopp's 100 year old rules on additiv~ O - ~(2)T ~ ity of atomic heat capacities17-19yield values within 2 cal of the above more reliable estimates. Thus at 298"K, rounded respectively, with values of the constants determined via off is 160 and 140 cal/deg mol for caffeine and theoleast squares. The ACo pz analytical equations in cal/deg phylline, respectively. These remarkably high positive mol are obtained directly from the temperature derivatives values show the large degree of structuring induced by of eq 1 and 2: these two molecules. Equations 3 and 4 also show that AC",,(caffeine) = - 2185 7.66T (3) ACopz for both systems increases sharply with temperature, contrary to the behavior generally observed for low molecuAC",,(theophylline) = - 1667 5.90T (4) lar weight aliphatic nonelectrolytes which show negative The difference in values of ACop, for the two xanthines temperature coefficients of AC",, (methanol excepteds). may be considered as the contribution to AC",, by a CH2 The lack of data for other systems and possible inflection group. In the midrange of measurement (298-303OK), eq 3 points over wider temperature spans preclude any satisfacand 4 show that this contribution is 10-15 cal/deg mol. Extory models a t this time.
+
+
+
+
cop,
+
+
The Journal of Physicai Chemistry, Vol. 79,No. 6, 1975
J. Bernhardt and H. Pauly
584
Acknowledgment. The authors wish to express their gratitude to Hendrik Tuinstra for help with the leastsquares calculations, and to the Long Beach California State University Foundation for financial assistance. References and Notes (1) W. S. Hoffman, "Biochemistry of Clinical Medicine," 4th ed, Year Book Medical Publishers, Chicago, Ill., 1970. (2) D. Guttman and T. Higuchi, J. Amer. Pharmacol. Ass., Sci. Ed., 46, 4 (1957). (3) J. H. Stern, J. A. Devore, S. L. Hansen, and 0. Yavuz, J. Phys. Chem., 78, 1922 (1974). (4) G. deStevens, "Diuretics, Chemistry and Pharmacology," Academic Press, New York, N.Y., 1963. (5) J. H. Stern and A. Hermann, J. Phys. Chem., 72, 364 (1968). (6) J. H. Stern and T. Swearingen, J. Phys. Chem., 74, 167 (1970). (7) J. H. Stern, 0. Yavuz, and T. Swearingen, J. Chem. Eng. Data, 17, 182 (1970).
(8) F. Franks in "Hydrogen-Bonded Solvent Systems," A. K. Covington and P. Jones, Ed., Taylor and Francis, London, 1968. (9) F. Franks in "Water, A Comprehensive Treatise," Vol. 2, F. Franks, Ed., Plenum Press, New York, N.Y., 1973. (IO) J. S. Sarma and J. C. Ahiuwalia, Chem. SOC. Rev. (London), 2, 203 (1973). (1 1) J. H. Stern and C. W. Anderson, J. Phys. Chem., 68, 2528 (1964). (12) D. Guttman and T. Higuchi, J. Pharm. Scb, 60, 1267 (1971). (13) A. L. Thakkar, L. G. Tensmeyer, and W. L. Williams, J. Pharm. Sci., 60, 1267 (1971). (14) J. Kirschbaum, J. Pharm. Sci., 62, 109 (1973). (15) R. D. Stiehier and H. M. Huffman, J. Amer. Chem. SOC.,57, 1741 (1935). (16) J. P. McCullough, H. L. Finke, J. F. Messerly, S. S. Todd, J. C. Klnchenloe, and G. Waddington, J. Phys. Chem., 61,1105 (1957). (17) H. Kopp, Ann. Chem. Pharm. Suppl., 3, 1, 289 (1864). (18) L. Paullng, "General Chemistry," 2nd ed, W. H. Freeman, San Francisco, Calif., 1959, p 638. (19) G. N. Lewis and M. Randall, "Thermodynamics," revised by K. S. Pitzer and L. Brewer, McGraw-Hill, New York, N.Y., 1961, p 58.
Partial Specific Volumes in Highly Concentrated Protein Solutions. I. Water-Bovine Serum Albumin and Water-Bovine Hemoglobin J. Bernhardt* and H. Pauly institut fur PhysikalischeundMedizinische Strahlenkunde der Universltat Erlangen-Nurnberg, 8520 Erlangen, West Germany (Received July 23, 1974; Revised Manuscript Received November 25, 1974)
Measurements of the specific volume of highly concentrated and salt-free solutions of bovine serum albumin (BSA) and bovine hemoglobin (Hb) a t 25' were carried out by using a digital densimeter. The protein mass fractions in the binary mixture were determined by measuring the dry weight with an accuracy of better than 0.01%. We found that the specific volumes of both solutions are not linear functions of the protein mass fraction. There exist very small deviations from linearity, more in BSA solutions than in H b s o h tions. The data were approximated by a power series. The partial specific volumes for water and hydrated proteins were determined with the aid of this power series. The partial specific volume of water increases with increasing protein mass fraction. The maximum increase for BSA solutions is about 0.4% (at 0.34 BSA mass fraction) and for H b solutions about 0.3% (at 0.44 H b mass fraction). According to this increase there is a decrease of the partial specific volume of the hydrated proteins. We found for the partial specific volume of BSA in infinitely diluted solution a value of V B S A ~= 0.73604 cm3 8-l and for H b vHbm = 0.75460 cm3 g-l.
I. Introduction Measurements of the specific volume of highly concentrated protein solutions were not of particular interest in the past. As only the limiting values upw of the partial specific volume of protein in infinitely diluted solutions were needed, measurements were made only with rather diluted protein solutions. Because of the increasing interest in the state of water in the living cell and in protein solutions of physiological concentrations measurements of the specific volume may now have a new bearing. However, until now there has been no report of a concentration dependence of the partial specific volumes in protein solution~.l-~ In order to understand better the behavior of water in the living cell, 'many experiments have been reported in scientific literature. As was resumed by Pauly,4 measurements of some properties of protein solutions, e.g., the dielectric constant and the electrical conductivity (Pauly and Schwan5), the heat of melting (Has1 and P a ~ l y ~ and , ~ ) the , The Journal ot Physical Chemistry, Voi. 79,No. 6, 1975
partial specific heat (Milbe@), have led to the result that approximately 0.2-0.4 g of water per gram of dry protein are bound in mixtures of protein and water, and the rest behaves like normal water. After Kratky et al.9 had developed the digital densimeter, it has become possible to measure easily and with sufficient accuracy the specific volumg of small amounts of highly viscous solutions. However in order t o detect changes of the partial specific volumes, the protein concentration of the solutions has to be determined with an accuracy of 0.01% or better. We determined the partial specific volume vw of water in bovine erythrocytes and stated a small, but significant increase of v, in erythrocytes.1° As we were not able to separate the influence of the proteins from that of the salts on the partial specific volume of water, we used salt-free protein solutions as simple models and measured their specific volume. The results given in this paper show that the removal of water from protein so-
