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Gadolinium and calcium binding to bovine serum albumin
J. inorg, nucl. Chem., 1976, Vol. 38, pp. 1415-1419. Pergamon Press. Printed in Great Britain
GADOLINIUM AND CALCIUM BINDING TO BOVINE SERUM ALBUMIN HERBERT B. SILBER* and JULIE ROSEN Division of Earth and Physical Sciences, University of Texas at San Antonio (UTSA), San Antonio, TX 78285, U.S.A. and Department of Chemistry, University of Maryland Baltimore County, Baltimore, MD 21228, U.S.A.
(First received 16 June 1975; in revisedform 25 September 1975) Abstract--The temperature jump relaxation technique is used to study the binding of calcium(II) and gadolinium(III) to bovine serum albumin at 7°C and an ionic strength of 0.2 (NaC10,). Both metal ions are predicted to bind to the -amino groups of the protein above pH = 8.2, where extensive metal ion hydrolysis takes place. For the reaction between GdOH2. and bovine serum albumin, the complex formation rate constant is (1.0_+0.1) x 10=M i sec-1 and the dissociation rate constant is (9.3-+2.1)sec ~. The corresponding calcium association reaction is too rapid to obtain rate constants. The complex formation rate constant is compared to other gadolinium systems to explain the relatively high complexation constant with bovine serum albumin.
INTRODUCTION THE use of lanthanide ions as a probe to determine calcium binding sites has been recently reviewed[l]. Although the interactions between calcium and the lanthanide ions with bovine serum albumin (BSA) have been investigated by differential spectroscopy [2, 3], electron spin relaxation[4], PMR[5] and by our earlier temperature jump relaxation measurements [6, 7], a complete description of these systems is not available. In the absence of binding metal ions, the temperature jump relaxation kinetics of BSA are described in terms of the proton transfer reactions of the imidazole (histidine) and e-amino (lysine) groups between pH 5.3 and 8.7 [6, 7]. The relaxation data after the addition of excess Ca(II) or Gd(III) ions are identical to the data for the apo-BSA solutions below pH 8.2. Our original interpretation of this observation is that neither cation binds to the histidine groups of BSA. Recently, another lanthanide ion, Nd(III), has been detected to bind to the imidazole groups of histidine at pH 7[8], and this may indicate that the temperature jump technique cannot detect this binding in a protein. The experimental data could not be fit to the appropriate relaxation equations assuming dimerization, which is known to exist between pH 3 and 3.5 [9], or by assuming a conformation change, which has been reported around pH 7 with a corresponding change in the histidine pK [10]. An ultrasonic relaxation investigation of conformation changes in aqueous solutions of BSA has demonstrated that the maximum effects occur below pH 4.3 and above pH 10.511]. Thus, these changes can be ignored in the pH range of this and the earlier temperature jump studies. At a pH in excess of 8.2, the relaxation data systematically differs from the apo-BSA solution data. The assumption that Gd 3÷ directly binds to one or more groups does not improve the fit of the data to the derived equations [7]. Thus, this study was initiated to determine the mechanisms by which the cations bind to the BSA. In
addition, no information is available concerning the kinetics of lanthanide complexation with large ligands, and this is of interest because of the current debate about whether lanthanide complexation occurs via a dissociative mechanism[12, 13]. EXPERIMENTAL
Stock solutions of BSA (Miles Laboratory, Kankanee, IL) were prepared by weight (molecular weight was assumed to be 65,000114]) and stored in the refrigerator for periods up to 1 week. The stock solutions of the metal ions were made from calcium nitrate (Baker, Analyzed) and from gadolinium oxide (Lindsay Rare Earths) dissolved in dilute perchloric acid. Chlorophenol red (pKI, = 6.08115]) or metacresol purple (pK~n= 8.15 at 0.2 ionic strength [6]) were used to monitor pH changes in the temperature jump experiments. All solutions were at an ionic strength of 0.2 with sodium perchlorate as the inert electrolyte. The kinetic measurements were carried out using a temperature jump spectrophotometer from Messanlagen Studiengesellschaft mbh (Gottingen, Germany). A Lauda Ultra Kryomat with R20 digital thermoregulator was used to insure thermal equilibrium within the temperature jump cell prior to the discharge of the 0.05 tzF capacitor (at 40kV) through the solution. All kinetic measurements were at a final temperature of 7°C. The pH of the solution was measured after the temperature jumps using a Coming 112 digital pH meter and a Corning combination micro electrode. The pH of the test solution was found to change after an appreciable number of jumps were made, probably due to decomposition of the BSA, but this problem was minimizedby the experimental procedure of only permitting a few discharges through the solution before replacing it with fresh solution. All calculations were carried out with a Wang 720 programmable calculator. RESULTS The relaxation data for the apo-BSA solutions and for the Gd(III)-BSA solutions below pH 8.2 can be explained by Reactions (1)-(6). k12
*H-BSA-lysH÷~
'BSA-IysH + + H ÷
(1)
k23
÷H-BSA-lysH++OH ~
*Address correspondence to this author in care of UTSA. 1415
k32
~BSA-lysI-I÷+H:O (2)
1416
HERBERT B. SILBER a n d JULIE ROSEN k34
BSA-IysH+~
'BSA-Iys + H +
(3)
k43 k45
BSA-lysH + + O H - ,
~BSA-lys + H20
i
(4)
to
k54
HIn = H + + In-
T
(5)
H20 = H + + OH-
(6)
o '_o
where +H-BSA-lysH + represents BSA with protonated imidiazole (histidine) groups (pKhis = 7'0) and protonated ~-amino (lysine) groups (pKIy~ = 9.8); where the loss of protons is represented by the removal of H + from the symbols; and HIn and In represent the protonated and ionic forms of the indicator[14, 16]. By assuming reactions (4)-(6) are in rapid equilibrium with (1)-(3), the relaxation equations are[7]: r - l O -1 = ks, +
2
200
~P° ° O/o
'
o
k32~
(7)
0 = [H +] - [ B S A - l y s H +] (2/3 - 1)1 y + K.(1 - 13)
(8)
/3 = ([BSA-lys] - TKIy,)/(7[H +] + 2[BSA-Iys])
(9)
y = 1 + [OH ]/[H +] + [In ]/(K,. + [H+])
(10)
q~ = [1 + Khis(1 -/3)/[H +] + Kh~s(1-2/3)[+H-BSA-lysH+]/y[H+y]/O
40 .0
i
(11)
where Kin is the acid dissociation constant for the respective indicators. Figure 1 is the apo-BSA relaxation data plotted according to eqns (1)-(11), indicating satisfactory agreement[& 7]. The relaxation and equilibrium data for the BSA solutions containing Gd(III) are presented in Table 1, and the Gd(III) solutions below pH 8.2 are included in Fig. 1. At higher pH's, the data would all fall below the line. The coupling of an additional reaction step involving direct Gd 3+ association to the e-amino groups leads to no improvement. This is unexpected since direct
2'00
I0-7~, M-I Fig. 1. Relaxation data for BSA solutions below pH 8.2. The line is the weighted least squares line and only randomly selected data at low ~ values are shown. O, apo-BSA; A, BSA solutions with Od(III). metal ion association is the mechanism consistent with the experimental data in other relaxation studies of lanthanide complexation systems carried out below pH 7117, 19]. At pH's greater than 6.5, the lanthanide ions are known to be hydrolyzed. If the reactive species is assumed to be GdOH 2+ (aq), two new reaction steps can be coupled to reactions (1)-(6): Gd 3++ H20 = GdOH 2++ H + GdOH~++BSA_lys,
(12)
~1 ,BSA_Iys_GdOH2+ '
(13)
ka
In the absence of BSA, at high pH where the GdOH 2+ species is formed, no relaxation is observed on our
Table 1. Equilibrium and relaxation data for the gadolinium-BSA solutions at 7°C and ionic strength 0.2 (NaCIO,) l0s [BSA] (M)
10" [cM] (M)
10s [H+] (M)
l0s [In ] (M)
10 3 z t (sec ')
104 10" [Gd3+] [GdOH2+] (M) (M)
A. Gadolinium-BSA solutions obeying reactions (1) through 3.17 0.101 105.0 1 8 - 3 C 13.2-+0.9 0.0960 3.17 0.0402 5 6 . 5 24.7C 11.5-+1.2 0.0391 6.34 0.101 36.5 28-8C 16.5-+2.4 0.0958 1.88 0.101 35.3 29.1C 19.3---1.9 0.0991 3.17 0.500 20.7 33.2C 8.8-+0.7 0 . 4 8 5 20.0 0.880 3.09 3.83M 17.3 -+ 1.2 0.692 12.0 0.880 2.83 4.10M 17.6-+2.0 0 . 7 4 7 11.1 8.80 2.36 2.35M 21.5 7.55 20.0 1.76 2.09 5.19M 19.6 -+0.6 1.33 40.0 0.880 0.973 8.64M 24.1 -+0.7 0 . 3 7 8
(6) 1.2×10 5 9.0×10 -5 0.00034 0.00037 0.00305 0.0291 0.0343 0.416 0.0830 0.0504
104 [BSA-IysGdOW +] (M)
106 [BSAlysH+l (M)
106 [BSAlys] (M)
(sec ')
107X (M)
_ 1.5×10 -~ 2.3x10 -7 7.7×10 7 2.7×10 -5 0.0226 0.0172 0.0795 0.0733 0.181
0-276 0.477 1.36 0.415 1.03 13.5 7.98 2.54 10.4 19.7
4.2×10 5 1.3x10-" 5.9×10-" 1.9×10 4 7.9×10-" 0.0692 0.0447 0.0170 0.0789 0.320
0.31 1.10 1.19 1.11 0.18 7.05 4.87 1.29 5.96 91.4
3.0×106 9.9x10 -6 3.2×10 -5 2.4×10 -~ 7.9×10 -~ 0-0217 0.0165 0.0282 0.0419 0.341
B. Gadolinium-BSA solutions obeying reactions (13) and (14) coupled to (1) through (6) 21.5 8.80 0.759 4.92M 26.3+0-7 6.09 1.04 0.203 0.831 35.8 8.80 0.619 5.44M 26.7-+ 1.4 4.98 1.05 0.342 1.14 14.3 8.80 0.548 5.75M 27.7-+1.7 6.22 1.47 0.139 0.291 17.9 8.80 0.497 5.99M 23-0+-0.8 5.88 1.54 0.175 0.319 7.16 8.80 0.392 6.57M 25.2-+1.1 6.19 2.05 0.0707 0.0761 17.9 8.80 0.315 7.06M 25.5---1.2 5.25 2.17 0.177 0.145
tX = ([GdOH2÷] + G[BSA-Iys])/A. $C = chlorophenol red, M = metacresol purple.
0.0174 0.0292 0.00842 0.0102 0.00308 0.00731
"r 1/A
13.6 21.1 27.2 27.5 48.6 77.3
0.550 0-848 1.45 1.85 3.96 6.57
1417
Gadolinium and calcium binding to bovine serum albumin instrument. This result is attributed to the hydrolysis reaction (12) being more rapid than can be observed by temperature jump techniques. With this assumption, a new relaxation expression can be derived for reactions (1)-(6) coupled to (12) and (13). r• '/A : ks([GdOH2~I+G[BSA-lysl)/A + k~ (14) A : F[GdOH 2~]/K~ + G(1 + [H+]/K,,)
(15)
I00
i--
~
t9 O~
50
v~
F = {2Kh + [H A]- ([BSA-lysH+I/[BSA-lys]) × (4Kh - 5[H+I)}/J
(16)
!
0
G : {[GdOH 2~] (1 + 5[BSA-lysH+]/[BSA-Iys])
I
i
L
50
I00
Fig. 2. Relaxation data for Gd(III) association to BSA above pH 8.2. The data at lower pH cannot be fit to the derived eqns (15)-(19). X is defined by eqns (14)-(17).
+ 3Kh [BSA-IysH+]/[H +] (17)
+ Khy[BSA-IysH+]/[BSA-Iys]}/J
DISCUSSION
J : 2([GdOH2~]+ 7[H+] + 7Kh) + [BSA-lysH +] (5 + 4Kh/[H+])
(18)
As before, y is defined by eqn (10) and K~ represents the hydrolysis constant for Gd 3÷, as written in eqn (12). The thermodynamic value of Kh is not known and the concentration quptient obtained by Moeller at low gadolinium concentrations, K, = 1.3 x 10-gM, is used[20]. The numerical value of the formation constant for the gadolinium-BSA complex is not known from independent measurements, but can be obtained from the relaxation data by an iterative method, since the formation constant, Ks = ks/ku. An additional modification must be made to the equilibrium data. These experiments cannot detect the dissociation of the carboxyl groups, or metal ion binding to the carboxyl groups. We assume that the carboxyl groups are totally ionized (pKa below 4 [14]) and that both Gd 3÷ and GdOH ~+ can bind to the carboxyl sites, with an apparent Gd(III) binding dissociation constant of 1.3 × 10-' [4]. Thus, in the presence of large excess of metal ion, the concentration of gadolinium that is available for binding at the E-amino sites would be approximately equal to:
available gadolinium = cM - cM. BSA/1.3
i
× 10-4
(19)
where cM and BSA are the stoichiometric concentrations of gadolinium and bovine serum albumin respectively. With these assumptions the equilibrium and relaxation data are calculated by eqns (14)--(18) by assuming an initial value of Ks. Then an analysis of eqn (14) is carried out using a weighted least squares. The ration of kt to kd is used to obtain a new KI and the cycle continued until the deviation in Ks was less than 1% from the assumed value. The data are shown in Fig. 2. The results of the calculations are ks = ( 1 ' 0 + 0 ' 1 ) × 108M - 1 sec 1, kd = (9'3 -+2.1) sec -~ and Kt = (1.1 -+0.4) x 107 M -~. The relaxation data for BSA solutions in the presence of calcium(II) below pH 8.2 are also indistinguishable from the data for the apo-BSA solutions under similar conditions. At pH's greater than 8.2, the relaxation present in the absence of calcium totally disappears, and we believe this indicates calcium binding to the E-amino groups, since the mechanism change occurs at the pH where the ~-amino groups start to ionize. In the absence of a measurable relaxation for the calcium ion complexation step, it is impossible to obtain kinetic data about the complexation step.
The disappearance of the relaxation signal in the BSA solutions in the presence of calcium above pH 8.2 is consistent with other temperature jump studies of calcium complexation, in which only a lower limit to the association constant can be assigned because the complexation reactions are too rapid to be observed [21]. The observation that calcium and gadolinium start to bind to BSA at the same pH indicates that they bind at the same sites. Saroff and Lewis have carried out equilibrium studies of calcium binding to serum albumin and they report an increase from 2.6 to 9.8 moles of calcium per mole of serum albumin as the pH increases from 8.0 to 10.0114], an effect also obtained in our kinetic measurements. Although gadolinium would normally be expected to bind at a specific site at a lower pH than the weaker binding calcium ion, the presence of a complexed ligand on the rare earth ion, such as OH- or even C10,-, can result in a decreased difference in binding ability between calcium and the lanthanide ions [22], consistent with the results of this study. At pH 9, Saroff[14] reports that the predominant binding involves a carboxylate ion imidazole and amino chelate. As the E-amino group ionization increases the number of calcium--chelate complexes increases, indicating that the binding dependsupon these e-amino groups. Based upon the kinetic similarity between calcium and gadolinium, we believe they exhibit the same binding characteristics in this system. Our kinetic results indicate that the binding of the metal ion to the E-amino groups is the slow step, consistent with Saroff's equilibrium results. If lanthanide complexation reactions follow a dissociative mechanism, then the measured complex formation constant, ks, for reaction with a biopolymer should be similar to that for simpler ligands. A summary of gadolinium complexation rate constants is presented in Table 2. The relaxation rate constants, reported as ks and kd, are obtained by temperature jump or pressure jump measurements using a mechanism similar to reaction (13). The ultrasonic relaxation rate constants, k34 and k43, refer to a modification of reaction (13): Gd3+(aq) + L" (aq) = Gd3+(H:O)xL" <
k34
,GdL ~ "(aq)
k43
(20) Step 12
Step III
where Gd3+(H20)xL "- represents an outer-sphere ionpair; Kc is the conductivity equilibrium constant defined
HERBERTB. SILBERand JUL1EROSEN
1418
Table2. Summaryof gaduliniumcomplexationdata in water Ligand, L
T (°C)
10-" kt Ionic strength (M-' sec-')
BSA Murexide[17] Murexide[18] Oxalate[19] Anthranilate[23] Anthranilate[18] Acetate[18]
7 12 12.5 25 9.0 12-5 12.5
0.2 (NaC104) 0.1 (KNO3) 0.2 (NaC10,) -0.2 (NaCIO4) 0.2 (NaCIO,) 0.2 (NaCIO4)
T (°C)
Ligand Nitrate[24] Sulfate[25] Sulfate[26] Sulfate[27]
25 25 25 25
k~ (sec ')
Kt
1.0-+0.1 0.52 0.9 0.46 0.43 0..59 0.74
9.3 -+2.1 4.3 x 103 -3.3 4.2 x 10" 4.7 × 10' --
1.I x 107 1.2 x 10' -1.4 x 107 1.3 × 103 1.3 x 103 --
Ultrasonicresults 10-s k3, Ionic strength (sec-~)
10-7 k,3 (sec-')
K~
17-+2 15 6.8 3.8
15-+2 4500 4500 4500
-----
by: Kc = Klz(1 + Kiu),
(21)
and Kl2 and Km are the equilibrium constants for steps 12 and III. If step 12 is rapid compared to step III, as is usually the case, then k/= Kl2k34.
(22)
Examination of Table 2 demonstrates that large variations in the magnitude of the association equilibrium constants have almost no effect upon the complex formation rate constant, but do effect the complex dissociation rate constant. Within experimental error, the ultrasonic complex formation rate constants are the same for the nitrate and sulfate complexes, a feature which is expected if complexation proceeds via a dissociative mechanism. The value of ky is greatest for the reaction with BSA, and this difference would be increased if the BSA reaction would have been investigated at the higher temperature utilized in the other studies. Eyring and Purdie have successfully accounted for differences in kt as a function of ionic strength and chelation[28, 29]. However, comparison of our BSA results with those of other systems at a constant ionic strength (0.2 M NaC10,) indicates no trend, and this effect cannot explain the BSA rate. If a chelate effect occurs, the rate-determining step is the formation of the second metal ion-ligand bond, resulting in a smaller rate constant compared to systems where the first cation-ligand bond is the slow step. This is contrary to the results of this study, indicating that a chelate effect does not occur in the Gf+-BSA system. We believe the relatively large complexation constant observed for gadolinium binding to BSA is due to the fact that the reactive species is the hydrolyzed GdOH2+(aq) ion rather than the free Gd3+(aq) ion. Although no other rate constants are present in the literature for the reactions of hydrolyzed lanthanide ions, the complexation constant for FeOH 2+ is significantly greater than that of Fe 3÷ with several ligands[40]. One cannot usually compare the reactions of a lanthanide ion directly with those of a d-type transition element, but we believe the conclusion to be valid in this case. The effect of hydroxide association to the plus-three lanthanide ion is to reduce the effective electrostatic charge of the metal ion, which
5_1.5 6.4 3.4 8.2
allows for a more rapid substitution rate for further complexation. Furthermore, previous studies of successive complexation steps in lanthanide-acetate systems show an enhanced formation rate for the bis-complex relative to the mono-complex[30]. Thus, after the GdOH 2+ complex is formed, further reaction with another ligand (BSA) would be accelerated. Either explanation results in the observed high complex formation for gadolinium association to BSA. Although this study cannot distinguish whether gadolinium complexation occurs via a dissociative mechanism, the results are not inconsistent with it. As a final point, this study was partially initiated to determine if a kinetic technique, such as temperature jump relaxation, can be utilized to probe calcium and lanthanide ion binding sites. Some success was achieved, notably with regard to binding at the E-amino (lys) groups. However, no binding has been detected at the imidazole sites (his), and this may occur in BSA. Thus, it is not clear if the effects of metal ion binding are sufficient to be detected through relaxation measurements. Further work using another protein with only one known binding site is suggested before temperature jump can be confidently utilized in determining metal ion binding sites. Acknowledgements--Acknowledgement is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research. This paper was presented at the 1lth Rare Earth Research Conference (Traverse City, Michigan, 1974). A portion of this work formed the undergraduate thesis of J. Rosen. REFERENCES
1. J. Reuben, Naturwissenschaflen 62, 172 (1975). 2. E. R. Bimbaum,J. E. Gomez and D. W. Darnall,J. Am. Chem. Soc. 92, 5287 (1970). 3. A."D. Sherry, E. R. Birnbaum and D. W. Darnall, J. Biol. Chem. 247, 3489 (1972). 4. J. Reuben, J. Phys. Chem. 75, 3164 (1971). 5. J. Reuben, Biochem. 10, 2834 (1972). 6. H. B. Silber, Biophys. Chem. 1, 300 (1974). 7. H. B. Silber and J. A. Rosen, FEBS Lett. 41, 43 (1974). 8. E. R. Birnbaum and D. W. Darnall, Bioinorg. Chem. 3, 15 (1973). 9. E. J. Williams and J. F. Foster, Jr. Am. Chem. Soc. 82, 3741 (1959).
Gadalinium and calcium binding to bovine serum albumin 10. B. J. M. Harmsen, S. H. DeBruin, L. H. M. Janssen, J. F. Rodriques De Miranda and G. A. J. Van Os, Biochem. 10, 3217 (1971). 11. L. W. Kessler and F. Duma, 3. Phys. Chem. 73, 4256 (1969). 12. H. B. Silber, J. C. S. Chem. Commun. 731 (1971). 13. M. M. Farrow and N. Purdie, J. Solution Chem. 2,513 (1973). 14. C. Tanford, S. A. Swenson and W. S. Shore, J. Am. Chem. Soc. 77, 6414 (1955); H. A. Saroff and M. S. Lewis, J. Phys. Chem. 67, 1211 (1%3). 15. M. Eigen and G. G. Hammes, J. Am. Chem. Soc. 82, 5951 (1%0). 16. The symbolism is somewhat different than in Refs. [6, 7]. 17. G. Geiger, Bet. Bunsenges. Physik. Chem. 69, 617 (1965). 18. H. B. Silber, R. D. Farina and J. H. Swinehart, Inorg. Chem. 8, 819 (1%9). 19. A. J. Graffeo and J. L. Bear, J. Inorg. Nucl. Chem. 30, 1577 (1968).
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20. T. Moeller, J. Phys. Chem. 50, 242 (1946). 21. R. G. Wilkins and M. Eigen, Advances in Chemistry Series (Edited by R. F. Gould), Vol. 49, pp. 55-80. Am. Chem. Soe. New York (1%5). 22. H. B. Silber, FEBS Lett. 41, 303 (1974). 23. H. B. Silber, Ph.D. Thesis, University of California, Davis (1967). 24. R. Garnsey and D. W. Ebdon, J. Am. Chem. Soc. 91, 50 (1%9). 25. D. P. Fay and N. Purdie, J. Phys. Chem. 74, 1160 (1970). 26. J. J. Grecsek, Ph.D. Thesis, University of Maryland, College Park (1%8). 27. J. Reidler and H. B. Silber, J. Phys. Chem. 77, 1275 (1973). 28. M. M. Farrow, N. Purdie and E. M. Eyring, Inorg. Chem. 13, 2024 (1974). 29. M. M. Farrow and N. Purdie, Inorg. Chem. 13, 2111 (1974). 30. M. Doyle and H. B. Silber, J. C. S. Chem. Commun. 1067 (1972).
GADOLINIUM AND CALCIUM BINDING TO BOVINE SERUM ALBUMIN HERBERT B. SILBER* and JULIE ROSEN Division of Earth and Physical Sciences, University of Texas at San Antonio (UTSA), San Antonio, TX 78285, U.S.A. and Department of Chemistry, University of Maryland Baltimore County, Baltimore, MD 21228, U.S.A.
(First received 16 June 1975; in revisedform 25 September 1975) Abstract--The temperature jump relaxation technique is used to study the binding of calcium(II) and gadolinium(III) to bovine serum albumin at 7°C and an ionic strength of 0.2 (NaC10,). Both metal ions are predicted to bind to the -amino groups of the protein above pH = 8.2, where extensive metal ion hydrolysis takes place. For the reaction between GdOH2. and bovine serum albumin, the complex formation rate constant is (1.0_+0.1) x 10=M i sec-1 and the dissociation rate constant is (9.3-+2.1)sec ~. The corresponding calcium association reaction is too rapid to obtain rate constants. The complex formation rate constant is compared to other gadolinium systems to explain the relatively high complexation constant with bovine serum albumin.
INTRODUCTION THE use of lanthanide ions as a probe to determine calcium binding sites has been recently reviewed[l]. Although the interactions between calcium and the lanthanide ions with bovine serum albumin (BSA) have been investigated by differential spectroscopy [2, 3], electron spin relaxation[4], PMR[5] and by our earlier temperature jump relaxation measurements [6, 7], a complete description of these systems is not available. In the absence of binding metal ions, the temperature jump relaxation kinetics of BSA are described in terms of the proton transfer reactions of the imidazole (histidine) and e-amino (lysine) groups between pH 5.3 and 8.7 [6, 7]. The relaxation data after the addition of excess Ca(II) or Gd(III) ions are identical to the data for the apo-BSA solutions below pH 8.2. Our original interpretation of this observation is that neither cation binds to the histidine groups of BSA. Recently, another lanthanide ion, Nd(III), has been detected to bind to the imidazole groups of histidine at pH 7[8], and this may indicate that the temperature jump technique cannot detect this binding in a protein. The experimental data could not be fit to the appropriate relaxation equations assuming dimerization, which is known to exist between pH 3 and 3.5 [9], or by assuming a conformation change, which has been reported around pH 7 with a corresponding change in the histidine pK [10]. An ultrasonic relaxation investigation of conformation changes in aqueous solutions of BSA has demonstrated that the maximum effects occur below pH 4.3 and above pH 10.511]. Thus, these changes can be ignored in the pH range of this and the earlier temperature jump studies. At a pH in excess of 8.2, the relaxation data systematically differs from the apo-BSA solution data. The assumption that Gd 3÷ directly binds to one or more groups does not improve the fit of the data to the derived equations [7]. Thus, this study was initiated to determine the mechanisms by which the cations bind to the BSA. In
addition, no information is available concerning the kinetics of lanthanide complexation with large ligands, and this is of interest because of the current debate about whether lanthanide complexation occurs via a dissociative mechanism[12, 13]. EXPERIMENTAL
Stock solutions of BSA (Miles Laboratory, Kankanee, IL) were prepared by weight (molecular weight was assumed to be 65,000114]) and stored in the refrigerator for periods up to 1 week. The stock solutions of the metal ions were made from calcium nitrate (Baker, Analyzed) and from gadolinium oxide (Lindsay Rare Earths) dissolved in dilute perchloric acid. Chlorophenol red (pKI, = 6.08115]) or metacresol purple (pK~n= 8.15 at 0.2 ionic strength [6]) were used to monitor pH changes in the temperature jump experiments. All solutions were at an ionic strength of 0.2 with sodium perchlorate as the inert electrolyte. The kinetic measurements were carried out using a temperature jump spectrophotometer from Messanlagen Studiengesellschaft mbh (Gottingen, Germany). A Lauda Ultra Kryomat with R20 digital thermoregulator was used to insure thermal equilibrium within the temperature jump cell prior to the discharge of the 0.05 tzF capacitor (at 40kV) through the solution. All kinetic measurements were at a final temperature of 7°C. The pH of the solution was measured after the temperature jumps using a Coming 112 digital pH meter and a Corning combination micro electrode. The pH of the test solution was found to change after an appreciable number of jumps were made, probably due to decomposition of the BSA, but this problem was minimizedby the experimental procedure of only permitting a few discharges through the solution before replacing it with fresh solution. All calculations were carried out with a Wang 720 programmable calculator. RESULTS The relaxation data for the apo-BSA solutions and for the Gd(III)-BSA solutions below pH 8.2 can be explained by Reactions (1)-(6). k12
*H-BSA-lysH÷~
'BSA-IysH + + H ÷
(1)
k23
÷H-BSA-lysH++OH ~
*Address correspondence to this author in care of UTSA. 1415
k32
~BSA-lysI-I÷+H:O (2)
1416
HERBERT B. SILBER a n d JULIE ROSEN k34
BSA-IysH+~
'BSA-Iys + H +
(3)
k43 k45
BSA-lysH + + O H - ,
~BSA-lys + H20
i
(4)
to
k54
HIn = H + + In-
T
(5)
H20 = H + + OH-
(6)
o '_o
where +H-BSA-lysH + represents BSA with protonated imidiazole (histidine) groups (pKhis = 7'0) and protonated ~-amino (lysine) groups (pKIy~ = 9.8); where the loss of protons is represented by the removal of H + from the symbols; and HIn and In represent the protonated and ionic forms of the indicator[14, 16]. By assuming reactions (4)-(6) are in rapid equilibrium with (1)-(3), the relaxation equations are[7]: r - l O -1 = ks, +
2
200
~P° ° O/o
'
o
k32~
(7)
0 = [H +] - [ B S A - l y s H +] (2/3 - 1)1 y + K.(1 - 13)
(8)
/3 = ([BSA-lys] - TKIy,)/(7[H +] + 2[BSA-Iys])
(9)
y = 1 + [OH ]/[H +] + [In ]/(K,. + [H+])
(10)
q~ = [1 + Khis(1 -/3)/[H +] + Kh~s(1-2/3)[+H-BSA-lysH+]/y[H+y]/O
40 .0
i
(11)
where Kin is the acid dissociation constant for the respective indicators. Figure 1 is the apo-BSA relaxation data plotted according to eqns (1)-(11), indicating satisfactory agreement[& 7]. The relaxation and equilibrium data for the BSA solutions containing Gd(III) are presented in Table 1, and the Gd(III) solutions below pH 8.2 are included in Fig. 1. At higher pH's, the data would all fall below the line. The coupling of an additional reaction step involving direct Gd 3+ association to the e-amino groups leads to no improvement. This is unexpected since direct
2'00
I0-7~, M-I Fig. 1. Relaxation data for BSA solutions below pH 8.2. The line is the weighted least squares line and only randomly selected data at low ~ values are shown. O, apo-BSA; A, BSA solutions with Od(III). metal ion association is the mechanism consistent with the experimental data in other relaxation studies of lanthanide complexation systems carried out below pH 7117, 19]. At pH's greater than 6.5, the lanthanide ions are known to be hydrolyzed. If the reactive species is assumed to be GdOH 2+ (aq), two new reaction steps can be coupled to reactions (1)-(6): Gd 3++ H20 = GdOH 2++ H + GdOH~++BSA_lys,
(12)
~1 ,BSA_Iys_GdOH2+ '
(13)
ka
In the absence of BSA, at high pH where the GdOH 2+ species is formed, no relaxation is observed on our
Table 1. Equilibrium and relaxation data for the gadolinium-BSA solutions at 7°C and ionic strength 0.2 (NaCIO,) l0s [BSA] (M)
10" [cM] (M)
10s [H+] (M)
l0s [In ] (M)
10 3 z t (sec ')
104 10" [Gd3+] [GdOH2+] (M) (M)
A. Gadolinium-BSA solutions obeying reactions (1) through 3.17 0.101 105.0 1 8 - 3 C 13.2-+0.9 0.0960 3.17 0.0402 5 6 . 5 24.7C 11.5-+1.2 0.0391 6.34 0.101 36.5 28-8C 16.5-+2.4 0.0958 1.88 0.101 35.3 29.1C 19.3---1.9 0.0991 3.17 0.500 20.7 33.2C 8.8-+0.7 0 . 4 8 5 20.0 0.880 3.09 3.83M 17.3 -+ 1.2 0.692 12.0 0.880 2.83 4.10M 17.6-+2.0 0 . 7 4 7 11.1 8.80 2.36 2.35M 21.5 7.55 20.0 1.76 2.09 5.19M 19.6 -+0.6 1.33 40.0 0.880 0.973 8.64M 24.1 -+0.7 0 . 3 7 8
(6) 1.2×10 5 9.0×10 -5 0.00034 0.00037 0.00305 0.0291 0.0343 0.416 0.0830 0.0504
104 [BSA-IysGdOW +] (M)
106 [BSAlysH+l (M)
106 [BSAlys] (M)
(sec ')
107X (M)
_ 1.5×10 -~ 2.3x10 -7 7.7×10 7 2.7×10 -5 0.0226 0.0172 0.0795 0.0733 0.181
0-276 0.477 1.36 0.415 1.03 13.5 7.98 2.54 10.4 19.7
4.2×10 5 1.3x10-" 5.9×10-" 1.9×10 4 7.9×10-" 0.0692 0.0447 0.0170 0.0789 0.320
0.31 1.10 1.19 1.11 0.18 7.05 4.87 1.29 5.96 91.4
3.0×106 9.9x10 -6 3.2×10 -5 2.4×10 -~ 7.9×10 -~ 0-0217 0.0165 0.0282 0.0419 0.341
B. Gadolinium-BSA solutions obeying reactions (13) and (14) coupled to (1) through (6) 21.5 8.80 0.759 4.92M 26.3+0-7 6.09 1.04 0.203 0.831 35.8 8.80 0.619 5.44M 26.7-+ 1.4 4.98 1.05 0.342 1.14 14.3 8.80 0.548 5.75M 27.7-+1.7 6.22 1.47 0.139 0.291 17.9 8.80 0.497 5.99M 23-0+-0.8 5.88 1.54 0.175 0.319 7.16 8.80 0.392 6.57M 25.2-+1.1 6.19 2.05 0.0707 0.0761 17.9 8.80 0.315 7.06M 25.5---1.2 5.25 2.17 0.177 0.145
tX = ([GdOH2÷] + G[BSA-Iys])/A. $C = chlorophenol red, M = metacresol purple.
0.0174 0.0292 0.00842 0.0102 0.00308 0.00731
"r 1/A
13.6 21.1 27.2 27.5 48.6 77.3
0.550 0-848 1.45 1.85 3.96 6.57
1417
Gadolinium and calcium binding to bovine serum albumin instrument. This result is attributed to the hydrolysis reaction (12) being more rapid than can be observed by temperature jump techniques. With this assumption, a new relaxation expression can be derived for reactions (1)-(6) coupled to (12) and (13). r• '/A : ks([GdOH2~I+G[BSA-lysl)/A + k~ (14) A : F[GdOH 2~]/K~ + G(1 + [H+]/K,,)
(15)
I00
i--
~
t9 O~
50
v~
F = {2Kh + [H A]- ([BSA-lysH+I/[BSA-lys]) × (4Kh - 5[H+I)}/J
(16)
!
0
G : {[GdOH 2~] (1 + 5[BSA-lysH+]/[BSA-Iys])
I
i
L
50
I00
Fig. 2. Relaxation data for Gd(III) association to BSA above pH 8.2. The data at lower pH cannot be fit to the derived eqns (15)-(19). X is defined by eqns (14)-(17).
+ 3Kh [BSA-IysH+]/[H +] (17)
+ Khy[BSA-IysH+]/[BSA-Iys]}/J
DISCUSSION
J : 2([GdOH2~]+ 7[H+] + 7Kh) + [BSA-lysH +] (5 + 4Kh/[H+])
(18)
As before, y is defined by eqn (10) and K~ represents the hydrolysis constant for Gd 3÷, as written in eqn (12). The thermodynamic value of Kh is not known and the concentration quptient obtained by Moeller at low gadolinium concentrations, K, = 1.3 x 10-gM, is used[20]. The numerical value of the formation constant for the gadolinium-BSA complex is not known from independent measurements, but can be obtained from the relaxation data by an iterative method, since the formation constant, Ks = ks/ku. An additional modification must be made to the equilibrium data. These experiments cannot detect the dissociation of the carboxyl groups, or metal ion binding to the carboxyl groups. We assume that the carboxyl groups are totally ionized (pKa below 4 [14]) and that both Gd 3÷ and GdOH ~+ can bind to the carboxyl sites, with an apparent Gd(III) binding dissociation constant of 1.3 × 10-' [4]. Thus, in the presence of large excess of metal ion, the concentration of gadolinium that is available for binding at the E-amino sites would be approximately equal to:
available gadolinium = cM - cM. BSA/1.3
i
× 10-4
(19)
where cM and BSA are the stoichiometric concentrations of gadolinium and bovine serum albumin respectively. With these assumptions the equilibrium and relaxation data are calculated by eqns (14)--(18) by assuming an initial value of Ks. Then an analysis of eqn (14) is carried out using a weighted least squares. The ration of kt to kd is used to obtain a new KI and the cycle continued until the deviation in Ks was less than 1% from the assumed value. The data are shown in Fig. 2. The results of the calculations are ks = ( 1 ' 0 + 0 ' 1 ) × 108M - 1 sec 1, kd = (9'3 -+2.1) sec -~ and Kt = (1.1 -+0.4) x 107 M -~. The relaxation data for BSA solutions in the presence of calcium(II) below pH 8.2 are also indistinguishable from the data for the apo-BSA solutions under similar conditions. At pH's greater than 8.2, the relaxation present in the absence of calcium totally disappears, and we believe this indicates calcium binding to the E-amino groups, since the mechanism change occurs at the pH where the ~-amino groups start to ionize. In the absence of a measurable relaxation for the calcium ion complexation step, it is impossible to obtain kinetic data about the complexation step.
The disappearance of the relaxation signal in the BSA solutions in the presence of calcium above pH 8.2 is consistent with other temperature jump studies of calcium complexation, in which only a lower limit to the association constant can be assigned because the complexation reactions are too rapid to be observed [21]. The observation that calcium and gadolinium start to bind to BSA at the same pH indicates that they bind at the same sites. Saroff and Lewis have carried out equilibrium studies of calcium binding to serum albumin and they report an increase from 2.6 to 9.8 moles of calcium per mole of serum albumin as the pH increases from 8.0 to 10.0114], an effect also obtained in our kinetic measurements. Although gadolinium would normally be expected to bind at a specific site at a lower pH than the weaker binding calcium ion, the presence of a complexed ligand on the rare earth ion, such as OH- or even C10,-, can result in a decreased difference in binding ability between calcium and the lanthanide ions [22], consistent with the results of this study. At pH 9, Saroff[14] reports that the predominant binding involves a carboxylate ion imidazole and amino chelate. As the E-amino group ionization increases the number of calcium--chelate complexes increases, indicating that the binding dependsupon these e-amino groups. Based upon the kinetic similarity between calcium and gadolinium, we believe they exhibit the same binding characteristics in this system. Our kinetic results indicate that the binding of the metal ion to the E-amino groups is the slow step, consistent with Saroff's equilibrium results. If lanthanide complexation reactions follow a dissociative mechanism, then the measured complex formation constant, ks, for reaction with a biopolymer should be similar to that for simpler ligands. A summary of gadolinium complexation rate constants is presented in Table 2. The relaxation rate constants, reported as ks and kd, are obtained by temperature jump or pressure jump measurements using a mechanism similar to reaction (13). The ultrasonic relaxation rate constants, k34 and k43, refer to a modification of reaction (13): Gd3+(aq) + L" (aq) = Gd3+(H:O)xL" <
k34
,GdL ~ "(aq)
k43
(20) Step 12
Step III
where Gd3+(H20)xL "- represents an outer-sphere ionpair; Kc is the conductivity equilibrium constant defined
HERBERTB. SILBERand JUL1EROSEN
1418
Table2. Summaryof gaduliniumcomplexationdata in water Ligand, L
T (°C)
10-" kt Ionic strength (M-' sec-')
BSA Murexide[17] Murexide[18] Oxalate[19] Anthranilate[23] Anthranilate[18] Acetate[18]
7 12 12.5 25 9.0 12-5 12.5
0.2 (NaC104) 0.1 (KNO3) 0.2 (NaC10,) -0.2 (NaCIO4) 0.2 (NaCIO,) 0.2 (NaCIO4)
T (°C)
Ligand Nitrate[24] Sulfate[25] Sulfate[26] Sulfate[27]
25 25 25 25
k~ (sec ')
Kt
1.0-+0.1 0.52 0.9 0.46 0.43 0..59 0.74
9.3 -+2.1 4.3 x 103 -3.3 4.2 x 10" 4.7 × 10' --
1.I x 107 1.2 x 10' -1.4 x 107 1.3 × 103 1.3 x 103 --
Ultrasonicresults 10-s k3, Ionic strength (sec-~)
10-7 k,3 (sec-')
K~
17-+2 15 6.8 3.8
15-+2 4500 4500 4500
-----
by: Kc = Klz(1 + Kiu),
(21)
and Kl2 and Km are the equilibrium constants for steps 12 and III. If step 12 is rapid compared to step III, as is usually the case, then k/= Kl2k34.
(22)
Examination of Table 2 demonstrates that large variations in the magnitude of the association equilibrium constants have almost no effect upon the complex formation rate constant, but do effect the complex dissociation rate constant. Within experimental error, the ultrasonic complex formation rate constants are the same for the nitrate and sulfate complexes, a feature which is expected if complexation proceeds via a dissociative mechanism. The value of ky is greatest for the reaction with BSA, and this difference would be increased if the BSA reaction would have been investigated at the higher temperature utilized in the other studies. Eyring and Purdie have successfully accounted for differences in kt as a function of ionic strength and chelation[28, 29]. However, comparison of our BSA results with those of other systems at a constant ionic strength (0.2 M NaC10,) indicates no trend, and this effect cannot explain the BSA rate. If a chelate effect occurs, the rate-determining step is the formation of the second metal ion-ligand bond, resulting in a smaller rate constant compared to systems where the first cation-ligand bond is the slow step. This is contrary to the results of this study, indicating that a chelate effect does not occur in the Gf+-BSA system. We believe the relatively large complexation constant observed for gadolinium binding to BSA is due to the fact that the reactive species is the hydrolyzed GdOH2+(aq) ion rather than the free Gd3+(aq) ion. Although no other rate constants are present in the literature for the reactions of hydrolyzed lanthanide ions, the complexation constant for FeOH 2+ is significantly greater than that of Fe 3÷ with several ligands[40]. One cannot usually compare the reactions of a lanthanide ion directly with those of a d-type transition element, but we believe the conclusion to be valid in this case. The effect of hydroxide association to the plus-three lanthanide ion is to reduce the effective electrostatic charge of the metal ion, which
5_1.5 6.4 3.4 8.2
allows for a more rapid substitution rate for further complexation. Furthermore, previous studies of successive complexation steps in lanthanide-acetate systems show an enhanced formation rate for the bis-complex relative to the mono-complex[30]. Thus, after the GdOH 2+ complex is formed, further reaction with another ligand (BSA) would be accelerated. Either explanation results in the observed high complex formation for gadolinium association to BSA. Although this study cannot distinguish whether gadolinium complexation occurs via a dissociative mechanism, the results are not inconsistent with it. As a final point, this study was partially initiated to determine if a kinetic technique, such as temperature jump relaxation, can be utilized to probe calcium and lanthanide ion binding sites. Some success was achieved, notably with regard to binding at the E-amino (lys) groups. However, no binding has been detected at the imidazole sites (his), and this may occur in BSA. Thus, it is not clear if the effects of metal ion binding are sufficient to be detected through relaxation measurements. Further work using another protein with only one known binding site is suggested before temperature jump can be confidently utilized in determining metal ion binding sites. Acknowledgements--Acknowledgement is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research. This paper was presented at the 1lth Rare Earth Research Conference (Traverse City, Michigan, 1974). A portion of this work formed the undergraduate thesis of J. Rosen. REFERENCES
1. J. Reuben, Naturwissenschaflen 62, 172 (1975). 2. E. R. Bimbaum,J. E. Gomez and D. W. Darnall,J. Am. Chem. Soc. 92, 5287 (1970). 3. A."D. Sherry, E. R. Birnbaum and D. W. Darnall, J. Biol. Chem. 247, 3489 (1972). 4. J. Reuben, J. Phys. Chem. 75, 3164 (1971). 5. J. Reuben, Biochem. 10, 2834 (1972). 6. H. B. Silber, Biophys. Chem. 1, 300 (1974). 7. H. B. Silber and J. A. Rosen, FEBS Lett. 41, 43 (1974). 8. E. R. Birnbaum and D. W. Darnall, Bioinorg. Chem. 3, 15 (1973). 9. E. J. Williams and J. F. Foster, Jr. Am. Chem. Soc. 82, 3741 (1959).
Gadalinium and calcium binding to bovine serum albumin 10. B. J. M. Harmsen, S. H. DeBruin, L. H. M. Janssen, J. F. Rodriques De Miranda and G. A. J. Van Os, Biochem. 10, 3217 (1971). 11. L. W. Kessler and F. Duma, 3. Phys. Chem. 73, 4256 (1969). 12. H. B. Silber, J. C. S. Chem. Commun. 731 (1971). 13. M. M. Farrow and N. Purdie, J. Solution Chem. 2,513 (1973). 14. C. Tanford, S. A. Swenson and W. S. Shore, J. Am. Chem. Soc. 77, 6414 (1955); H. A. Saroff and M. S. Lewis, J. Phys. Chem. 67, 1211 (1%3). 15. M. Eigen and G. G. Hammes, J. Am. Chem. Soc. 82, 5951 (1%0). 16. The symbolism is somewhat different than in Refs. [6, 7]. 17. G. Geiger, Bet. Bunsenges. Physik. Chem. 69, 617 (1965). 18. H. B. Silber, R. D. Farina and J. H. Swinehart, Inorg. Chem. 8, 819 (1%9). 19. A. J. Graffeo and J. L. Bear, J. Inorg. Nucl. Chem. 30, 1577 (1968).
1419
20. T. Moeller, J. Phys. Chem. 50, 242 (1946). 21. R. G. Wilkins and M. Eigen, Advances in Chemistry Series (Edited by R. F. Gould), Vol. 49, pp. 55-80. Am. Chem. Soe. New York (1%5). 22. H. B. Silber, FEBS Lett. 41, 303 (1974). 23. H. B. Silber, Ph.D. Thesis, University of California, Davis (1967). 24. R. Garnsey and D. W. Ebdon, J. Am. Chem. Soc. 91, 50 (1%9). 25. D. P. Fay and N. Purdie, J. Phys. Chem. 74, 1160 (1970). 26. J. J. Grecsek, Ph.D. Thesis, University of Maryland, College Park (1%8). 27. J. Reidler and H. B. Silber, J. Phys. Chem. 77, 1275 (1973). 28. M. M. Farrow, N. Purdie and E. M. Eyring, Inorg. Chem. 13, 2024 (1974). 29. M. M. Farrow and N. Purdie, Inorg. Chem. 13, 2111 (1974). 30. M. Doyle and H. B. Silber, J. C. S. Chem. Commun. 1067 (1972).