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Complex formation in the blood of Helix pomatia
.
Corn/~. Bwdwn, P/ry.~iol. Vol. 74A. No. I. pp. 165 to 168. 19X3 Printed in Great Britain.
COMPLEX
FORMATION IN THE BLOOD OF HELIX POMATZA E.
Institut
fuer Anorganische
R.
and B. M.
WERNER
und Analytische (Recriwd
Chemie. 9 March
RODE
Universitaet
Innsbruck,
Austria
1982)
1. Complex formation between calcium, haemocyanin from Helix porn&a, D-lactate and succinate has been investigated by means of potentiometric titrations using iterative curve fitting procedures. 2. Strong complexes containing more than two constituents could not be detected. 3. The method proved to be useful to investigate the protein-calcium complex formation. 4. The model thus derived, using acid-base and complex formation equilibria, agrees within the concentration range of 2@40 mM Ca” with an empirical formula of pH-dependence of calcium binding by Helix potnutia haemocyanin.
Abstract
INTRODLICTION
The investigations presented here were stimulated by an investigation concerning responses of Helix pomatia to anoxia (Table 1) (Wieser, 1981). Despite the increase of the solute concentration, caused mainly by calcium. D-lactate and succinate, the total solute activity decreases considerably from 10 to 40 hr of anoxia (Table 1). This behaviour was interpreted as complex formation. Therefore the system calcium. u-lactate, succinate and haemocyanin from He/ix porn&a was investigated by potentiometric titrations to reveal the composition and stability of possibly formed complex species. MATERIALS
AND
METHODS
Mrthod Acid- base titration is a frequently used method for analyzing complex formation. A system of equations can be constructed consisting of the ion products of water and all possible acid-base and complex formation equilibria, the mass balances of each constituent and the charge balance. A set of pK-values, which is chosen arbitrarily, is varied to obtain the best fit to the titration curves. The dpK, values defined in this work denote the significance of the thus derived constants pKi, in the sense that a change in the constant pK, by dpK, leads to an increase of FQ by a factor of 2. FQ is defined by: X Z (titrant added-titrant computed)2, the summation being performed over all points of a titration and over all titration curves. Due to the large number of groups per molecule titrated in the case of a protein some simplifications are inevitable. The most common one is to assign a single pK-value to all chemically equivalent constituents (e.g. to all histidine side chains). To determine the concentration of these constituents, their concentration is varied in addition to the set of pK-values when searching for the best fit to the titration curve. Scope and limits of this method of analyzing titration curves of proteins alone are discussed by Marini et ul. ( 1974). After having obtained a description of the protein titration curves in this way, the complex formation of the protein with other components of the solution can be treated analogous to the complex formation of small molecules.
Experimental All solutions were prepared using distilled water freed from COZ by boiling under N, atmosphere. The ionic strength of all solutions was adjusted to 0.2 by adding NaCl. All titrations were performed at 20 f 0.3 ‘C in N, atmosphere. Haemocyanin was prepared from the haemolymph as follows: after centrifugation (2 hr. 50,OOOg) and chromatography (diethylaminoethylcellulose equilibrated with triethanolamine-HCl, pH = 7.5) the haemocyanin fraction was dialyzed 24 hr against distilled water. The protein concentration was measured at 278 nm in 0.1 M sodium borate buffer pH = 9.2. using the specific extinction coefficient A (1%. 1 cm) = 14.16 (Heirwegh et al., 1961). Succinic acid (“puriss. p.a.“, Fluka), Na-L-lactate (“purum”, Fluka) and Li-o-lactate (“reinst”, Serva) were used. The pH-measurements were carried out by an lngold microelectrode 104051393 and a Schott pH-meter CG-803. The calibration was performed using standard buffer solutions (Merck). The concentration of free calcium ions was determined by a F-2000 Selektrode (Radiometer). All calculations were performed at the CDC Cyber-74 computer of the University of Innsbruck. RESULTS
1. Complexes qf calcium with lactate and succinate These well known values have been determined to test method and apparatus. In the concentration range occurring in Helix porn&a haemolymph (Table 1) only 1: 1 complexes of calcium(2 +) with lactate( -) and succinate(2 - ) are formed in detectable quantities, and therefore. no complexes consisting of all three components could be detected in this concentration range. The summarized results are as following: succinic acid : pK, = 4.00 + 0.03 C4.00 (Campi,
1963)]
pK, = 5.25 & 0.04 [5.28 (Campi. 1963)J calcium complex Ca(2 + ) + Suc(2 - ) - SucCa(0) pK3 = -0.82 [ - 1 (Schubert
+ 0.15
& Lindenbaum,
- 1.2 (Campi. 1963).
1952).
.
E. R. WERNER and B. M. RODE
166
Table 1. Averageconcentrationsof main componentsand total solute activity of haemolymphof Helix pomatia during anoxia accordingto Wieser(1981) Duration of anoxia (hr) 0 5
10 15 20 30 40
Na+
Cl-
60 70 70 70 70 70 70
50 50 65 65 65 65 65
Concentration(mM) K+ Mg*+ Ca2+ 4 5 6 8 9 12 14
10 10
17
20 20 20 17 16
26 32 41 60 73
- 1.2 (Cannan 8c Kibrik, 1938)
8
D-Lac-
Succ*-
0 25 35 42 47 55 60
0 8 14 20 26 37 45
Other anions Approximate Total solute mainly total concentration activity HCO; (mOsm) (estimated) bW 40 40 40 40 40 40 40
170 225 275 300 320 355 380
160 220 270 265 260 240 220
presented by Engelborghs et al. (1976). Their description consists of only one group, the histidine side -2 (Topp & Davies, 1940)] chains, with a pK of 7.2 and a concentration of lactic acid: pKi = 3.65 f 0.05 C3.739 (Cannan & 310 + 5pmol/g haemocyanin. They pointed out, however, that a number of other groups could be Kibrik, 1938)] present. calcium complex Ca(2+) + Lac( -) -+ LacCa( +) The curve fitting procedure used in our work is pK2 = -0.84 + 0.28 especially advantageous in this case, because pK[ -0.93 (Cannan & Kibrik, 1938) - 1.46 (Davies. values and group concentrations of overlapping ionizations can be extracted also when only a part of the 1938) titration curve is experimentally obtainable (Marini et -0.8 (Schubert & Lindenbaum, 19.52). al., 1974). Both amino acids, whose side chains are expected to have a pK-value of about 9, namely tyro- 1.42 (Davies & Monk, 1954). sine and cysteine, are found in haemocyanin of Helix - 0.90 (Veerbeek & Thun, 1965) pomatia (Gruber, 1966). (b) Calcium complexes of Helix pomatia haemocya-0.93 (Lepri & Desideri, 1973) nin. Calcium complex formation has been tested in -0.87 (Lepri & Desideri, 1973) the range of 2@70mmol calcium/l and at a protein - 1.74 (Malecki et al., 1978)] concentration of about 10 g/l. The titration curves can be fitted sufficiently on the basis of the following 2. Complexes containing Helix pomatia haemocyanin model : (a) Acid-base titrations of haemocyanin. The protein Ca(2+) + B(O)-+ BCa(2+) solution (pH = 8) was titrated to pH = 7 with HCI followed by back-titration with NaOH to pH = 9.5. pK = - 1.45 + 0.10. Outside this region the titration curve becomes time The result that complex formation occurs at group dependent for both calcium free and calcium contain- “B” and not at group “A” can be demonstrated by the ing solutions. The titration curves of He/ix pomatia titration curves (Figs 1 & 2). Titration curves of the haemocyanin can be fitted sufficiently using the folprotein with calcium differ remarkably from the titralowing model : tion curves of the protein itself in the region where mainly group “B” is titrated bH < 8.3, the amounts BH(+)--*B(O) + H(+) of species BH(+) and B(0) show strong pH-depenpK, = 7.40 f 0.02 dence]. In the part where mainly group “A” is titrated copt = 282 f 4pmol/g protein [pH > 8.3, the amounts of species AH and A(-) show strong pH-dependence] this differe ce becomes AH(O)-+A(-) + H(+) L small and is insignificant at pH > 9. pK, = 9.05 &-0.15 Literature values: R. F. Burton (Burton, 1972) has studied the binding of calcium to Helix pomatia haecopt = 180 + 25 pmol/g protein. mocyanin by ultracentrifugation and equilibrium di,&j&-value of about 7 (group “B”) is to be expected alysis.He summarized his results in the empirical forfti histidine side chains and free u-amino groups, a mula (I) pK-value of about 9 (group “AH”) for cysteine and v = CO.73(pH - 4) - 0.0433 (pH)‘]Ca, tyrosine side chains (Tanford, 1962). As free u-amino Ch (1) (groups are not found in Helix pomatia haemocyanin Cal, + 4.70(9.445 - pH) ’ (Cox et al., 1972), group “B” can be assigned to histidine side chains only (Engelborghs et al., 1976). Vc, being the amount of calcium bound (mmol/g haeLiterature values: An analysis of the titration mocyanin), Ca, being the concentration of free calcurvesof-Helix pomatia haemocyanin from pH 6 to 9 cium in the supernatant or dialysate (mmol/Icg water). using the.‘model of Linderstrom and Lang has been For comparison Vc, can be calculated using the pK-
Complex formation in the blood of He/k po~atia _
(b)
I/
I
2
I
3
4
pm01
%
Fig. 1. (a) Species distribution as a function of pH ~ without Ca(2+) --- with 55.5 mM Ca(Z+) haemocyanin concentration = 11.7 g/l, B(total) = 100%. (b) Acid titration curves of Helix porn&a haemocyanin ~ without Ca(Z+) .-.- with 25.0 mM Ca(2f) . with 37.0 mM Ca(2+) - - - with 55.5 mM Cd@ + ) haemocyaninconcentration = 11.7 g/l,
values of this work as follows:
Combining the above equations leads to (II) nK,Ca(Z+) L = 1 + K;‘H(+) + K,Ca(2+)’
B(tot) = B(0) + BH(+) + BCa(2f) K, = W)H(+) a BH(+) BCa(2+) Kc = B(O)Ca(2+) Vc, = BCa(2-t) n
B(tot)
n being the amount of group “B” in mmol/g protein,
all other quantities being concentrations in mol/l.
A comparison of the two formulas (I), (II) is shown in Table 2. Outside the region of 20-40 mM (Ca(2+) the thus calculated values for V,-, differ increasingly. (c) Ternary and quarternary complexes.TO fit the titration curves of mixtures of calcium, D-lactate, succinate and haemocyanin in the concentration range shown in Table 1, no complexes except those already described were required. Therefore strong complexes that could explain the osmotic behaviour of Helix
1 %
(II)
2
4
3
I
5
6
pmoC
Fig. 2. Base back titration analogous to Fig. I. (a) Species distribution as a function of pH ~ without Ca(2+) - -- with 36.9 mM Ca(2+) haemocyanin concentration = 7.77 g/l, B(tota1) = 100%. (b) Base titration curves of Helix pomatia haemocyanin without Ca(2+) .-.- with 16.6 mM Ca(2+) . with 24.6 mM Ca(2 +) - - - with 36.9 mM Ca ‘+ haemocyanin concentration = 7.77 g/l.
Table 2. Vc, values calculated by both formulas (I) and (II) using IZ = 0.282 mmol/g, pK, = 7.40, pK, = - 1.45 Ca(2+)
.
E. R. WERNERand B. M. RODE
168
= Ca, (mM) 29.6 31.5 33.8 37.0
I/,,(I) (mmoW
PH 7.21 7.45 7.68 8.02
0.068 0.088 0.106 0.126
I/,,UI) (mmok) 0.069 0.090 0.109 0.129
porn&a haemolymph during anoxia (Table 1) should not be formed by the constituents investigated here. According to our results, the following amounts of calcium are bound after 40 hr of anoxia [haemocyanin concentration = 20 g/l, HCO; = 27 mM, pK,(HCO;) = 6.34, pK,(HCO,Ca(+)] = -O.Sl(Sillen, 1964), calcium(tot) = 73 mM, lactate = 60 mM, succinate = 45 mM, pH = 6.90): 1.7% to haemocyanin; 6.5% to HCO; ; 18.6% to lactate and 13.9% to succinate.
constituents of the haemolymph other than those considered in this work are responsible for the observed osmotic behaviour. Acknowledgement-Financial support by the Austrian Federal Ministry for Science and Research Erl. Zl. 18 854/6-lo/81 is gratefully acknowledged. REFERENCES BURTON R. F. (1972) The binding of alkaline earth ions by the haemocyanin of Helix pomatia. Camp. Biochem. Physiol. 41A. 555-565. CAMPI E. (1963) Complessi di ioni metallici con gli acidi tatronico, malice. malonico e succinico. Ann. Chim. 53(-2).
9&l
16.
CANNAN R. K. & KIBRIK A. (1938) Complex formation between carboxylic acids and divalent metal cations. J. Am. them. Sot. 60, 23142320. Cox J., WITTERSR. & LONT~ER. (1972) Quarternary structure of He/ix porn&a haemocyanins as determined by alkali treatment and succinylation. Int. J. Biochem. 3, 283-293.
DISCUSSION Some uncertainty arises from the comparatively narrow pH range of the titration. Any complex formation that does not change the titration in this pH region can not be detected by the method used here. In order to reduce this uncertainty, the concentration of free calcium(2+) has been measured by means of an ion selective electrode. In all cases, however, the measured concentration of free calcium(2+) agreed with the corresponding values calculated from the potentiometric titrations within the range of experimental errors. The pK-values and the concentration of the titrated groups of the protein found by the curve fitting procedure depends to some extent on the initial values used when this procedure is started (cf: Marini et al., 1974). It was tested carefully, therefore, whether the occurrence of group “A” is realistic or a methodical artefact. It could be proved, however, that the titration curves cannot be fitted by any means assuming only one titrated group. The result, that calcium ions bind to the histidine side chains (group “I?‘) agrees with the common concepts of calcium binding to proteins (e.g. Saroff & Lewis, 1963) The description of calcium binding to Helix pomatia haemocyanin as being discussed in this work can be regarded surely as a very simple model with respect to the complex nature of this protein. The comparison with the results of other methods shows, however, that this description is satisfactory for the concentration range studied. Our investigation also shows that any more detailed model cannot be achieved within the reproducibility and accuracy of our experiments. One can imagine two reasons why complexes explaining the anomalous behaviour of the osmotic pressure in Helix pomatia blood during anoxia were not found. One reason might be, that a possible change of the quarternary and tertiary structure of the protein during the isolation procedure destroyed the capability of the protein to form some strong specific comnlexes. Another nossible reason would be that -----r~-~~~~ ~~ I
DAVIES C. W. (1938) The extent of dissociation of salts in water, part VI. Some calcium salts of organic acids. J. them. Sot. 277-281. DAVIES P. B. & MONK C. B. (1954) E.m.f. studies of electrolytic dissociation, part 6, Some lactates in water. Trans. Faraday
Sot. 50, 132-136.
ENGELBORGHSY., DEBRUIN S. H. & LONTIE R. (1976) Differential hydrogen ion titrations of the hi&dine residues of Helix pomatia haemocyanin. Biophys. Chem. 4, 343-348.
GRUBERM. (1966) Structure and function of Helix pomatia haemocyanin. Physiol. Biochem. Haemocyanins, Symp. 1966, (Edited by GH~RETTI F.), pp. 49-59. Academic Press, London. HEIRWEGH K., BORGINON H. & LONTIE R. (1961) Separation and absorption spectra of CI-and j-hemocyanin of Helix
pomatia.
Biochem.
biophys.
Acta
48, 517-526.
LEPRI L. & DESIDERI P. G. (1973) Influence of complex formation on the chromatographic behaviour of inorganic ions on sodium carboxvmethvlcellulose and Dowex 50-X4 (Na+) J. Chromat. 8;1, 1551164. MALECKI F.. STAROSCIKR. & ZIETEK J. (1978) Zur KomDlexbildung in Calciumprlparaten. Pharmake 33, H2;3, 98-99.
MARINI M. A., MARTIN C. J., BERGERR. L. & FORLANI L. (1974) A proposed solution for the determination of ionization constants of sets of ionizing groups in proteins. Biopolymers 13, 891-902. SAROFFH. A. & LEWIS M. S. (1963) The binding of calcium ions to serum albumin. J. phys. Chem. 67, 1211-1216. SCHUBERTJ. & LINDENBAUM A. (1952) Stabilitv of alkaline earth organic acid complexes as measured by ion exchange. .I. Am. them. Sot. 74, 3529-3531. SILLEN L. G. (1964) Stability constants of metal ion complexes, compiled by Lars Gunnar Sillen. Special Publication No. 17, p. 137. The Chemical Society, London. TANFORD C. (i962) The interpretation of hydrogen ion titration curves of proteins. Adv. Protein Chem. 17. 69-165. TOPP N. E. & DAVIES C. W. (1940) The extent of dissociation of salts in water, part IX. Calcium and barium salts of dicarboxylic acids. J. them. Sot. 87-93. VEERBEEKF. and THUN H. (1965) The stability constants of alkaline earth lactate and a-hydroxyisobutyrate complexes. Anal. chim. Acta 33, 378-383. WIESERW. (1981) Responses of Helix pomatia to anoxia: Changes of solute activity and other properties of the haemolymph. J. camp. Physiol. 141, 503-509.
Corn/~. Bwdwn, P/ry.~iol. Vol. 74A. No. I. pp. 165 to 168. 19X3 Printed in Great Britain.
COMPLEX
FORMATION IN THE BLOOD OF HELIX POMATZA E.
Institut
fuer Anorganische
R.
and B. M.
WERNER
und Analytische (Recriwd
Chemie. 9 March
RODE
Universitaet
Innsbruck,
Austria
1982)
1. Complex formation between calcium, haemocyanin from Helix porn&a, D-lactate and succinate has been investigated by means of potentiometric titrations using iterative curve fitting procedures. 2. Strong complexes containing more than two constituents could not be detected. 3. The method proved to be useful to investigate the protein-calcium complex formation. 4. The model thus derived, using acid-base and complex formation equilibria, agrees within the concentration range of 2@40 mM Ca” with an empirical formula of pH-dependence of calcium binding by Helix potnutia haemocyanin.
Abstract
INTRODLICTION
The investigations presented here were stimulated by an investigation concerning responses of Helix pomatia to anoxia (Table 1) (Wieser, 1981). Despite the increase of the solute concentration, caused mainly by calcium. D-lactate and succinate, the total solute activity decreases considerably from 10 to 40 hr of anoxia (Table 1). This behaviour was interpreted as complex formation. Therefore the system calcium. u-lactate, succinate and haemocyanin from He/ix porn&a was investigated by potentiometric titrations to reveal the composition and stability of possibly formed complex species. MATERIALS
AND
METHODS
Mrthod Acid- base titration is a frequently used method for analyzing complex formation. A system of equations can be constructed consisting of the ion products of water and all possible acid-base and complex formation equilibria, the mass balances of each constituent and the charge balance. A set of pK-values, which is chosen arbitrarily, is varied to obtain the best fit to the titration curves. The dpK, values defined in this work denote the significance of the thus derived constants pKi, in the sense that a change in the constant pK, by dpK, leads to an increase of FQ by a factor of 2. FQ is defined by: X Z (titrant added-titrant computed)2, the summation being performed over all points of a titration and over all titration curves. Due to the large number of groups per molecule titrated in the case of a protein some simplifications are inevitable. The most common one is to assign a single pK-value to all chemically equivalent constituents (e.g. to all histidine side chains). To determine the concentration of these constituents, their concentration is varied in addition to the set of pK-values when searching for the best fit to the titration curve. Scope and limits of this method of analyzing titration curves of proteins alone are discussed by Marini et ul. ( 1974). After having obtained a description of the protein titration curves in this way, the complex formation of the protein with other components of the solution can be treated analogous to the complex formation of small molecules.
Experimental All solutions were prepared using distilled water freed from COZ by boiling under N, atmosphere. The ionic strength of all solutions was adjusted to 0.2 by adding NaCl. All titrations were performed at 20 f 0.3 ‘C in N, atmosphere. Haemocyanin was prepared from the haemolymph as follows: after centrifugation (2 hr. 50,OOOg) and chromatography (diethylaminoethylcellulose equilibrated with triethanolamine-HCl, pH = 7.5) the haemocyanin fraction was dialyzed 24 hr against distilled water. The protein concentration was measured at 278 nm in 0.1 M sodium borate buffer pH = 9.2. using the specific extinction coefficient A (1%. 1 cm) = 14.16 (Heirwegh et al., 1961). Succinic acid (“puriss. p.a.“, Fluka), Na-L-lactate (“purum”, Fluka) and Li-o-lactate (“reinst”, Serva) were used. The pH-measurements were carried out by an lngold microelectrode 104051393 and a Schott pH-meter CG-803. The calibration was performed using standard buffer solutions (Merck). The concentration of free calcium ions was determined by a F-2000 Selektrode (Radiometer). All calculations were performed at the CDC Cyber-74 computer of the University of Innsbruck. RESULTS
1. Complexes qf calcium with lactate and succinate These well known values have been determined to test method and apparatus. In the concentration range occurring in Helix porn&a haemolymph (Table 1) only 1: 1 complexes of calcium(2 +) with lactate( -) and succinate(2 - ) are formed in detectable quantities, and therefore. no complexes consisting of all three components could be detected in this concentration range. The summarized results are as following: succinic acid : pK, = 4.00 + 0.03 C4.00 (Campi,
1963)]
pK, = 5.25 & 0.04 [5.28 (Campi. 1963)J calcium complex Ca(2 + ) + Suc(2 - ) - SucCa(0) pK3 = -0.82 [ - 1 (Schubert
+ 0.15
& Lindenbaum,
- 1.2 (Campi. 1963).
1952).
.
E. R. WERNER and B. M. RODE
166
Table 1. Averageconcentrationsof main componentsand total solute activity of haemolymphof Helix pomatia during anoxia accordingto Wieser(1981) Duration of anoxia (hr) 0 5
10 15 20 30 40
Na+
Cl-
60 70 70 70 70 70 70
50 50 65 65 65 65 65
Concentration(mM) K+ Mg*+ Ca2+ 4 5 6 8 9 12 14
10 10
17
20 20 20 17 16
26 32 41 60 73
- 1.2 (Cannan 8c Kibrik, 1938)
8
D-Lac-
Succ*-
0 25 35 42 47 55 60
0 8 14 20 26 37 45
Other anions Approximate Total solute mainly total concentration activity HCO; (mOsm) (estimated) bW 40 40 40 40 40 40 40
170 225 275 300 320 355 380
160 220 270 265 260 240 220
presented by Engelborghs et al. (1976). Their description consists of only one group, the histidine side -2 (Topp & Davies, 1940)] chains, with a pK of 7.2 and a concentration of lactic acid: pKi = 3.65 f 0.05 C3.739 (Cannan & 310 + 5pmol/g haemocyanin. They pointed out, however, that a number of other groups could be Kibrik, 1938)] present. calcium complex Ca(2+) + Lac( -) -+ LacCa( +) The curve fitting procedure used in our work is pK2 = -0.84 + 0.28 especially advantageous in this case, because pK[ -0.93 (Cannan & Kibrik, 1938) - 1.46 (Davies. values and group concentrations of overlapping ionizations can be extracted also when only a part of the 1938) titration curve is experimentally obtainable (Marini et -0.8 (Schubert & Lindenbaum, 19.52). al., 1974). Both amino acids, whose side chains are expected to have a pK-value of about 9, namely tyro- 1.42 (Davies & Monk, 1954). sine and cysteine, are found in haemocyanin of Helix - 0.90 (Veerbeek & Thun, 1965) pomatia (Gruber, 1966). (b) Calcium complexes of Helix pomatia haemocya-0.93 (Lepri & Desideri, 1973) nin. Calcium complex formation has been tested in -0.87 (Lepri & Desideri, 1973) the range of 2@70mmol calcium/l and at a protein - 1.74 (Malecki et al., 1978)] concentration of about 10 g/l. The titration curves can be fitted sufficiently on the basis of the following 2. Complexes containing Helix pomatia haemocyanin model : (a) Acid-base titrations of haemocyanin. The protein Ca(2+) + B(O)-+ BCa(2+) solution (pH = 8) was titrated to pH = 7 with HCI followed by back-titration with NaOH to pH = 9.5. pK = - 1.45 + 0.10. Outside this region the titration curve becomes time The result that complex formation occurs at group dependent for both calcium free and calcium contain- “B” and not at group “A” can be demonstrated by the ing solutions. The titration curves of He/ix pomatia titration curves (Figs 1 & 2). Titration curves of the haemocyanin can be fitted sufficiently using the folprotein with calcium differ remarkably from the titralowing model : tion curves of the protein itself in the region where mainly group “B” is titrated bH < 8.3, the amounts BH(+)--*B(O) + H(+) of species BH(+) and B(0) show strong pH-depenpK, = 7.40 f 0.02 dence]. In the part where mainly group “A” is titrated copt = 282 f 4pmol/g protein [pH > 8.3, the amounts of species AH and A(-) show strong pH-dependence] this differe ce becomes AH(O)-+A(-) + H(+) L small and is insignificant at pH > 9. pK, = 9.05 &-0.15 Literature values: R. F. Burton (Burton, 1972) has studied the binding of calcium to Helix pomatia haecopt = 180 + 25 pmol/g protein. mocyanin by ultracentrifugation and equilibrium di,&j&-value of about 7 (group “B”) is to be expected alysis.He summarized his results in the empirical forfti histidine side chains and free u-amino groups, a mula (I) pK-value of about 9 (group “AH”) for cysteine and v = CO.73(pH - 4) - 0.0433 (pH)‘]Ca, tyrosine side chains (Tanford, 1962). As free u-amino Ch (1) (groups are not found in Helix pomatia haemocyanin Cal, + 4.70(9.445 - pH) ’ (Cox et al., 1972), group “B” can be assigned to histidine side chains only (Engelborghs et al., 1976). Vc, being the amount of calcium bound (mmol/g haeLiterature values: An analysis of the titration mocyanin), Ca, being the concentration of free calcurvesof-Helix pomatia haemocyanin from pH 6 to 9 cium in the supernatant or dialysate (mmol/Icg water). using the.‘model of Linderstrom and Lang has been For comparison Vc, can be calculated using the pK-
Complex formation in the blood of He/k po~atia _
(b)
I/
I
2
I
3
4
pm01
%
Fig. 1. (a) Species distribution as a function of pH ~ without Ca(2+) --- with 55.5 mM Ca(Z+) haemocyanin concentration = 11.7 g/l, B(total) = 100%. (b) Acid titration curves of Helix porn&a haemocyanin ~ without Ca(Z+) .-.- with 25.0 mM Ca(2f) . with 37.0 mM Ca(2+) - - - with 55.5 mM Cd@ + ) haemocyaninconcentration = 11.7 g/l,
values of this work as follows:
Combining the above equations leads to (II) nK,Ca(Z+) L = 1 + K;‘H(+) + K,Ca(2+)’
B(tot) = B(0) + BH(+) + BCa(2f) K, = W)H(+) a BH(+) BCa(2+) Kc = B(O)Ca(2+) Vc, = BCa(2-t) n
B(tot)
n being the amount of group “B” in mmol/g protein,
all other quantities being concentrations in mol/l.
A comparison of the two formulas (I), (II) is shown in Table 2. Outside the region of 20-40 mM (Ca(2+) the thus calculated values for V,-, differ increasingly. (c) Ternary and quarternary complexes.TO fit the titration curves of mixtures of calcium, D-lactate, succinate and haemocyanin in the concentration range shown in Table 1, no complexes except those already described were required. Therefore strong complexes that could explain the osmotic behaviour of Helix
1 %
(II)
2
4
3
I
5
6
pmoC
Fig. 2. Base back titration analogous to Fig. I. (a) Species distribution as a function of pH ~ without Ca(2+) - -- with 36.9 mM Ca(2+) haemocyanin concentration = 7.77 g/l, B(tota1) = 100%. (b) Base titration curves of Helix pomatia haemocyanin without Ca(2+) .-.- with 16.6 mM Ca(2+) . with 24.6 mM Ca(2 +) - - - with 36.9 mM Ca ‘+ haemocyanin concentration = 7.77 g/l.
Table 2. Vc, values calculated by both formulas (I) and (II) using IZ = 0.282 mmol/g, pK, = 7.40, pK, = - 1.45 Ca(2+)
.
E. R. WERNERand B. M. RODE
168
= Ca, (mM) 29.6 31.5 33.8 37.0
I/,,(I) (mmoW
PH 7.21 7.45 7.68 8.02
0.068 0.088 0.106 0.126
I/,,UI) (mmok) 0.069 0.090 0.109 0.129
porn&a haemolymph during anoxia (Table 1) should not be formed by the constituents investigated here. According to our results, the following amounts of calcium are bound after 40 hr of anoxia [haemocyanin concentration = 20 g/l, HCO; = 27 mM, pK,(HCO;) = 6.34, pK,(HCO,Ca(+)] = -O.Sl(Sillen, 1964), calcium(tot) = 73 mM, lactate = 60 mM, succinate = 45 mM, pH = 6.90): 1.7% to haemocyanin; 6.5% to HCO; ; 18.6% to lactate and 13.9% to succinate.
constituents of the haemolymph other than those considered in this work are responsible for the observed osmotic behaviour. Acknowledgement-Financial support by the Austrian Federal Ministry for Science and Research Erl. Zl. 18 854/6-lo/81 is gratefully acknowledged. REFERENCES BURTON R. F. (1972) The binding of alkaline earth ions by the haemocyanin of Helix pomatia. Camp. Biochem. Physiol. 41A. 555-565. CAMPI E. (1963) Complessi di ioni metallici con gli acidi tatronico, malice. malonico e succinico. Ann. Chim. 53(-2).
9&l
16.
CANNAN R. K. & KIBRIK A. (1938) Complex formation between carboxylic acids and divalent metal cations. J. Am. them. Sot. 60, 23142320. Cox J., WITTERSR. & LONT~ER. (1972) Quarternary structure of He/ix porn&a haemocyanins as determined by alkali treatment and succinylation. Int. J. Biochem. 3, 283-293.
DISCUSSION Some uncertainty arises from the comparatively narrow pH range of the titration. Any complex formation that does not change the titration in this pH region can not be detected by the method used here. In order to reduce this uncertainty, the concentration of free calcium(2+) has been measured by means of an ion selective electrode. In all cases, however, the measured concentration of free calcium(2+) agreed with the corresponding values calculated from the potentiometric titrations within the range of experimental errors. The pK-values and the concentration of the titrated groups of the protein found by the curve fitting procedure depends to some extent on the initial values used when this procedure is started (cf: Marini et al., 1974). It was tested carefully, therefore, whether the occurrence of group “A” is realistic or a methodical artefact. It could be proved, however, that the titration curves cannot be fitted by any means assuming only one titrated group. The result, that calcium ions bind to the histidine side chains (group “I?‘) agrees with the common concepts of calcium binding to proteins (e.g. Saroff & Lewis, 1963) The description of calcium binding to Helix pomatia haemocyanin as being discussed in this work can be regarded surely as a very simple model with respect to the complex nature of this protein. The comparison with the results of other methods shows, however, that this description is satisfactory for the concentration range studied. Our investigation also shows that any more detailed model cannot be achieved within the reproducibility and accuracy of our experiments. One can imagine two reasons why complexes explaining the anomalous behaviour of the osmotic pressure in Helix pomatia blood during anoxia were not found. One reason might be, that a possible change of the quarternary and tertiary structure of the protein during the isolation procedure destroyed the capability of the protein to form some strong specific comnlexes. Another nossible reason would be that -----r~-~~~~ ~~ I
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MARINI M. A., MARTIN C. J., BERGERR. L. & FORLANI L. (1974) A proposed solution for the determination of ionization constants of sets of ionizing groups in proteins. Biopolymers 13, 891-902. SAROFFH. A. & LEWIS M. S. (1963) The binding of calcium ions to serum albumin. J. phys. Chem. 67, 1211-1216. SCHUBERTJ. & LINDENBAUM A. (1952) Stabilitv of alkaline earth organic acid complexes as measured by ion exchange. .I. Am. them. Sot. 74, 3529-3531. SILLEN L. G. (1964) Stability constants of metal ion complexes, compiled by Lars Gunnar Sillen. Special Publication No. 17, p. 137. The Chemical Society, London. TANFORD C. (i962) The interpretation of hydrogen ion titration curves of proteins. Adv. Protein Chem. 17. 69-165. TOPP N. E. & DAVIES C. W. (1940) The extent of dissociation of salts in water, part IX. Calcium and barium salts of dicarboxylic acids. J. them. Sot. 87-93. VEERBEEKF. and THUN H. (1965) The stability constants of alkaline earth lactate and a-hydroxyisobutyrate complexes. Anal. chim. Acta 33, 378-383. WIESERW. (1981) Responses of Helix pomatia to anoxia: Changes of solute activity and other properties of the haemolymph. J. camp. Physiol. 141, 503-509.