The determination of the stability constant for calcium EGTA

321 Biochimica et Biophysica Acta, 451 (1976) 321--325 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 21434

THE DETERMINATION OF THE STABILITY CONSTANT FOR CALCIUMEGTA

JEFFREY D. OWEN Department of Physiology, University of Utah, Salt Lake City, Utah 84132 (U.S.A.) (Received June 21st, 1976)

Summary The stability constant for calcium-EGTA, KCaEGTA , was determined in imidazole, Tris-maleate and phosphate buffered-solutions. The value for KCaEGTA was found to be independent of the buffer solution, in contrast with earlier reports (Ogawa, Y. (1968) J. Biochem. 64, 225; Godt, R.E. (1974) J. Gen. Physiol. 63, 722). Using a newly developed Ca 2+-selective electrode (Brown, H.M., Pemberton, J.P. and Owen, J.D. (1976) Anal. Chim. Acta, in the press) the Ca2+ activities in solutions of different [ CaEGTA] / [EGTA] ratio were found to fit a calibration plot of mV vs. log calcium activity using the KCaEGTA value of Schwarzenbach, G., Senn, H. and Anderegg, G. ((1957) Helv. Chim. Acta 40, 1886).

Ca 2* concentration changes occur in several important physiological processes and the highly selective calcium chelating agent, ethyleneglycol bis03aminoethylether)-N,N'-tetraacetic acid (EGTA), is frequently used to regulate the ionized concentration of calcium, [Ca2+]. In order to calculate Ca2+ concentration accurately, the correct stability constant for calcium-EGTA, KCaEGTA, is necessary.

The most recent determinations of KCaEGTA have almost always reported values which are lower than the earlier reported value of Schwarzenbach et al. [1]. The purpose of this paper is to show with a new Ca2 +-selective electrode that in some of the recent determinations of KCaEGTA , the measured amount of Ca 2+ was greater than was previously calculated, which would tend to yield an erroneously low value for KCaEGTA. The apparent or conditional stability constant for CaEGTA2: K~aEGTA , which is dependent upon pH, is Abbreviations: EGTA, ethyleneglycol bis(~-aminoethylether)-N,N'-tetraacetic acid; t-HDOPP, di[p-(1,1,3,3-tetramethylbutyl)phenyl]phosphoric acid; NTA, rdtrilotriacetic acid.

322

[CaEGTA] Ca 2+ + EGTAtotal ~ CaEGTA 2-

K~agO'rA = [~a2=~][EC, TAtOt~]

(])

where gGTAtota 1 = [EGTA 4-] + [EGTA 3-] + [EGTA 2-] + [EGTA-] + [EGTA] and with calcium-EGTA the predominant EGTA species which binds to Ca 2+ is [EGTA4-]. Rewriting Eqn. 1 yields [CaEGTA]

[Ca 2+] =

(2)

[EGTAtotal]KCaEGTA Eqn. 2 shows that Ca 2 + is dependent upon the concentration of the bound ligand (CaEGTA) and the unbound ligand (EGTAtotal). Eqn. 2 also shows K~aEGTA can be easily determined by measuring Ca 2+ concentration at known concentrations of bound and unbound ligand. "The relationship between ~he apparent and the "true" stability constant, KCaEGTA

2-, is

KCaEOTA 2-

' 2 - (1 + K, [it +] + K~ Ks [H+] 2 + K~K2Ka [H+] 3 + KCaEGTA

g, K2KaK4 [H+Y

(3)

where KI--K4 represent the stability constants for HEGTA 3-, H: EGTA 2-, HaEGTA-, and H4EGTA [2, 12]. We measured Ca 2 + concentration with a calcium-selective microelectrode, as described previously [3--5]. The electrode electroactive materials, di-[p(1,1,3,3-tetramethylbutyl)phenyl] phosphoric acid (t-HDOPP) and the calcium chelate of t-HDOPP, were dissolved in di-(n-octylphenyl)phosphoric acid and combined with polyvinylchloride to form a calcium-selective membrane in the tip of a pyrex glass microelectrode. The calibration plot of an electrode is shown in Fig. 1. The Ca 2+ concentration in the various solutions was calculated according to the stability constants for calcium-nitrilo-triacetic acid (NTA) [6] and calcium-ethylenediaminetetraacidic acid (EDTA) [1]. 70

® Ca CI 2

0 CaZ'(NTA) e CaZ'(EDTA)

30 •

CoZ'(EGTA)

~,-"

-~0

•

mV

j/c~ ./

/

/

-r~3 @ / @

-90 / z

150 8

7

~

2

I

pCo

Fig. 1. Calibration curve of calcium-selective m i c r o e l e c t r o d e in 20 mM i m i d a z o l e - b u f f e r e d s o l u t i o n s at p H 7.25. All s o l u t i o n s c o n t a i n e d 2 0 0 m M KC1. See t e x t for the e x p l a n a t i o n for the t w o E G T A points a b o v e the calibration curve at pCa 5 . 1 8 and 4 . 1 8 .

323

The stability constant for calcium-EGTA that we found which gave Ca 2÷ values which fit the calibration curve in Fig. 1 was the value originally proposed by Schwarzenbach et al. [1] and not the lower KCaEGTA proposed recently by several investigators [ 7--10]. If a lower calcium-EGTA stability constant were used to calculate the Ca 2 ÷ in the various EGTA solutions, then these points would be shifted to higher Ca 2÷ concentrations (to approximately four times more concentrated) and they would n o t fit on the calibration curve. It should be pointed o u t that the determination of KCaEGTA by this procedure is independent of the stability constants for NTA and EDTA, since the Ca 2+ from the EGTA solutions fall on the calibration curve initiated with pCa 1--3 solutions made with CaC12. Also, the calibration curve of these electrodes deviates less from linearity in the pCa 7--9 range with less KC1 added to the solutions which indicates the stability constant used for calcium-EDTA is correct. When similar solutions were prepared with Tris, histidine, Tris-maleate or phosphate (EGTA solutions only in this case}, instead of imidazole, the calcium electrode readings were also similar. Table I compares the KCaEGTA determined by the different procedures. It is interesting to note that the low KCaEGTA value determined with the chelex partition procedure was done at Ca 2+ values between 10 -6 and 10 -3 M [9--10]. We found EGTA gives erroneously high Ca 2+ concentrations in this concentration range, which would account for a low KCaEGTA , as shown by the electrode mV readings at pCa = 4.18 and 5.18 which are above the calibration curve in Fig. 1. This follows since the buffer capacity of EGTA is probably overwhelmed at the high ratio (approx. 102--10 s at pCa 6--3) of bound to u n b o u n d ligand. It is difficult to compare our data with the calciummurexide indicator experiments of Ogawa since no raw data was given [9]. Ogawa's dual beam technique was similar to Ebashi's [11], so Ogawa must have been looking at extremely small (approx. 10 -~ ) absorbance changes in the micromolar calcium region. Ogawa also determined KCaEGTA in solutions with and without 10 mM MgC12 and found no significant difference in KCaEGTA values [8]. This would indicate the limit of sensitivity of Ogawa's techniques since the Ca 2÷ concentration would be slightly higher in solutions containing MgC12, as shown in Fig. 2. Fig. 2 represents the theoretical effect of magnesium on Ca 2+ concentration using the calculations described previously 1,0

o.s

y

[EGTA]+o*2'0.6 0.4

I0 mM Mg,rotm

02 0

8

[

l

L I

i~lll

7

I

I

I

I

[ l III

6

I

I

J--/

llliJ

5

pCa Fig. 2. The theoretical e f f e c t of I 0 m M MgCI~ of pCa with [ E G T A ] t o t a l = 1 m M a n d p H ~ 6.8. The small

l o g d i v i s i o n s o n t h e abscissa are f o r e a s y c o n v e r s i o n s f r o m p C a

(-log

[Ca2+])

to

[Ca2+].

324

o

~o~

~'~

"0

+

~

~

m

m~

g

e

~ ,

N

g

325 (Owen, J.D. and Brown, H.M., unpublished results). Ebashi [7] also had 10 mM MgCI: in his calcium-EGTA solutions which would raise Ca2+ concentration, but this would not be enough to completely account for the low KCaEGTA determined by this procedure (see Fig. 2). Recently, Dipolo et al. [ 14] determined KCaEGTAspectrophotometrically with arsenazo III. Their value of 101°"96 is essentially the same as the Schwarzenbach group's earlier value of 1011.00. The fact that Dipolo et al. obtained the same value for KCaEGTA as Schwarzenbach et al., but in a buffer system not containing phosphate, suggests that phosphate does not appreciably bind calcium. The binding of phosphate would lower Ca 2+ concentration, which would increase the value of KCaEGTA [15, 16]. The significance of the higher KCaEGTA reported here than the value previously used by some investigators in muscle research [ 13] suggests the possibility that the amount of ionized calcium involved in the muscle contractionrelaxation cycle is about four times lower than some previous estimates. This work was supported in part by NIH Grant EY 00762 from the National Eye Institute awarded to Dr. H. Mack Brown. References 1 Schwarzenbach, G., Senn, H. and Anderegg, G. (1957) Helv. Chim. Acta 40, 1886--1900 2 Bje1~mm, J., Schwarzenbach, G. and Sillen, L.G. (1957) Stability Constants, Part I: Organic Ligands, pp. 76, 90, The Chemical Society, London 3 0 w e n , J.D., Brown, H.M. and Pemberton, J.P. (1976) Biophys. J. 16, 84a 4 Brown, H.M., Pembezton, J.P. and Owen, J.D. (1976) Anal. Chim. Acta, in the press 5 0 w e n , J.D., Brown, H.M. and Pemberton, J.P. (1976) Anal. Chim. Acta, submitted for publication 6 Ringbom, A. (1963) Complexatinn in Analytical Chemistry, Chemical Analysis, Vol. 16, p. 334, Interscience Publications, New York 7 Ebashi, S. (1961) J, Biochem. 50, 236--244 8 0 g a w a , Y. (1968) J. Biochem. 64, 255--257 9 Briggs, F.N. and Fleishman, M. (1965) J. Gen. Physiol. 49, 131--149 10 Godt, R.E. (1974) J. Gen. Physiol. 63, 722--739 11 Ohnishi0 T. and Ebashi, S. (1963) J. Biochem. 54, 506m511 12 Portzehl, H., Caldwell, P.C. and Ruegg, J.C. (1964) Biochim.Biophys. Acta 79, 581--591 13 Ebashl, S. and Endo, M. (1968) in Progress in Biophysics and Molecular Biology (Butler, J.A.V. and Noble, D., eds.), VoL 18, pp. 123k183, Pergamon Press, London 14 Dipolo, R., Requena, J., Brinley, Jr., F.J., Mullins, L.J., Scarpa, A. and Tiffert, T. (1976) J. Gen Physiol. 67, 433--467 15 Smith, R.M. and Alberty, R.A. (1956) J. Am. Chem. Soc. 78, 2376--2320 16 Simons, T.J.B. (1976) J. Physiol. 256, 209--225