Interaction of human fibrinogen with divalent metal chlorides

Interaction of human fibrinogen with divalent metal chlorides Hiroshi Maeda, Takaaki Kishi and Shoichi Ikeda Department of Chemistry, Faculty of Science, Naooya University, Chikusa-ku, Naooya, Japan

Susumu Sasaki Department of Physiology, Fujita-Gakuen University School of Medicine, Toyoake, Aichi, Japan

Kyoji Kito Meito Sangyo Company Ltd, Nagoya, Japan

(Received 1 November 1982; revised 27 November 1982) Precipitation of human fibrinooen in 0.15 M NaCI occurred at pH 7.4 (Tris-H CI buffer) when ZnC12, CuCI2, NiCI 2, or CoCl 2 were added beyond their respective critical concentrations. The critical concentrations were about 4 x l O -5 M ZnCI2, 6×10 -5 M CuCl2, 3 x l O -4 M NiCI 2 and 1 × 1 0 - 3 M CoCI 2. At pH 5.8 2-(Nmorpholino)-ethane sulphonic acid buffer, the critical concentrations were found only for CuCl 2 and ZnCl2, and were about 3 x 10 -5 and 3 x IO-a M, respectively. CaCl 2 and MgCI 2 were not effective up to 1 × 10 -2 and 2 × 10 -2 M at pH 7.4 and 5.8, respectively. At pH 7.4, precipitation was better in 0.015 M NaCl than in 0.15 M NaCl for both CuCl2 and ZnCl2. Erttle or no conformational chanoe was indicated on bindino Cu 2 ÷ ions. The fluorescence of tryptophan was quenched only by CuCl 2, while other metal ions (ZnCl 2, NiCl2, CoCl 2 and CaCl 2) were ineffective as quenchers. Keywords: Globulins; fibrinogen; precipitation; divalent metal ions

Introduction

Experimental

The interaction of fibrinogen with C a 2 + ions has been studied extensively I - 4 since Ca 2 + ions play an important role in the conversion of fibrinogen to fibrin 5. The stability of bovine fibrinogen against heat or acid denaturation increases on binding Ca 2÷ ions 1. Generally, binding Ca 2÷ ions does not induce any appreciable conformational change when examined by circular dichroism (c.d.) 1. The binding sites for Ca 2÷ ions are rather heterogeneous; the affinities of two or three sites are stronger, while those of others are weaker 1'2 However, a cobalt-fibrinogen complex, which contains two to four Co 2÷ ions, is known to be non-clottable 7's. Hydrodynamic data suggest a more compact structure for cobalt-fibrinogen than for normal fibrinogen 7. The helical content of cobalt-fibrinogen is higher than that of normal fibrinogen as suggested by c.d. data a. The interaction of fibrinogen with other divalent ions has been less extensively studied. Recently, a study on the interaction of bovine fibrinogen with metal ions was performed 9, concerned with the turbidity of solutions at a fixed pH and ionic strength. In the present work, the interaction of human fibrinogen with divalent ions such as Cu 2 + and Zn 2 ÷ ions has been studied using turbidity measurements, c.d., and fluorescence, at different values of pH and ionic strength. The effects of buffers on the interaction of fibrinogen with metal ions are also examined.

Human fibrinogen (Kabi, grade L) was dissolved in 0.15 M NaC1, dialysed against 0.15 M NaC1 and subsequently freeze-dried. Tris(hydroxymethyl)aminomethane(Tris) and 2-(N-morpholino)-ethane sulphonic acid(MES) were purchased from Nakarai Chemicals Ltd (Kyoto, Japan). Reagent grade CuCI2"6H20, ZnCIE, NiCIz'6H20, MgCI2"6H20 were purchased from Nakarai Chemicals Ltd (Kyoto). Reagent grade COC12.6H20 and CaClE'4H20 (suprapur) were from Merck. The protein solutions were prepared each day by dissolving the freezedried material in either Tris-HCl (pH7.4) or MES (pH 5.8) buffer, containing 0.15 M or 0.015 M NaCI. The protein concentration ( g d l - 1) was calculated by assuming the absorbance at 280 nm (1 cm) of a 1 g dl- 1 solution to be 16.0. Ultraviolet and visible absorption spectra and turbidity of the solutions were measured on a Shimadzu UV-200 spectrophotometer using a cell of 1 cm path length. Measurements of the turbidity were carried out mostly at 400nm by adding various amounts (0.014).1 cm 3) of a metal chloride solution to the protein solution (4 cm a) in a cuvette. The protein concentrations were 0.11 g dl-1 or 0.023-0.026gd1-1. The turbidity was measured 2 or 3 min after each addition of metal chloride solution. Circular dichroism (c.d.) measurements were performed on a Jasco J-40 A circular dichrograph using cells of 1 and 2 mm path lengths. The protein concentration was about 0.022 g d l - 1. The inner walls of the quartz cell were found to be hydrophobic after contact with fibrinogen solutions, due to adsorption of fibrinogen. The cell was washed with

Part of the present work was presented at the 34th Symposium on Colloid Chemistry,Japan, October 1981 0141-8130/83/030159q)4503.00 O 1983 Butterworth & Co. (Publishers) Ltd

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the buffers was examined and will be described later. Values of the critical concentration at pH 5.8 are considerably different from those at pH 7.4. They are 3-4 x 10- 5 M for CuC12 and 3 x 10 -4 M for ZnCl 2. The effects of CuC12 at pH 5.8 are approximately similar to those at pH 7.4 or are slightly enhanced. However, the effects of ZnC12, NiCI 2 and CoC12 are markedly reduced at pH 5.8. CaC12 and MgC12 are not effective when their concentrations are below 2 x 10- 2 M. " Different critical concentrations ofZnC12 at the two pH values suggests that the histidine residues of fibrinogen are the probable binding sites for Zn 2 + ions. The isoelectric point of bovine fibrinogen is 5.5 in 20% urea ~°. Precipitation is greater at pH 5.8 than at pH 7.4, since fibrinogen has a lower net charge at pH 5.8 than at pH 7.4. However, critical concentrations are lower at pH 7.4 than at pH 5.8 for all metal chlorides other than CuC1 z. The pH dependence of the critical concentration suggests that the precipitation caused by metal ion binding is not simply related to a decrease of net charges. Precipitation at low protein concentration by CuCI2 and ZnClz at 0.015 M NaCI is compared with that at 0.15 M NaC1 in Figure 2. Precipitation occurs at lower metal chloride concentrations for both CuC12 and ZnCIz at 0.015 M NaC1. The results indicate that the binding of Cu 2÷ and Zn 2+ ions becomes stronger as the ionic strength is lowered. However, fibrinogen itself is less soluble at an ionic strength of 0.015 M than at 0.15 M due to the salting-in effect 11. It is likely, therefore, that precipitation of fibrinogen is subject to both effects ot ionic strength on the binding and the solubility.

Circular dichroism and fluorescence ethanol-3 N HC1, 1 M Tris, 1 M N a O H , and/or conc. H 2 S O 4 , until the blank c.d. spectra (cell + buffer) showed no adsorbed fibrinogen. Fluorescence of the solution was measured on a Hitachi 610-10S spectrofluorimeter with a 1 x 1 cm cell. The protein concentrations were 6.6q5.8 x 10-3 and 0.026 g dl- 1.

C.d. spectra of fibrinogen were measured in the presence of various amounts of ZnC12 and CuC1 z at pH 7.4, to see whether a conformational change occurred prior to precipitation. The c.d. spectra showed that the or-helix was the only secondary structure significantly present in fibrinogen 5. When CuC12 was added, the residue ellipticity at 208 nm, [011o8, did not change significantly at low CuCI/concentrations and decreased in magnitude as the critical concentration was approached. This change oc-

Results

Precipitation of fibrinogen by divalent metals Precipitation of fibrinogen occurred on addition of various metal chlorides. The absorbance (turbidity) at 400nm was measured at pH 7.4 and 5.8. The turbidity increased rather abruptly after the metal concentration (CM) reached a certain value. Accordingly, a critical concentration for the precipitation could be defined. Measured turbidities were not stationary values but the critical concentration did not depend appreciably on the interval of the measurements. The turbidity curves obtained at pH 7.4 and 5.8 in 0.15 M NaCI are shown in Fioures la and Ib, respectively. Values of the critical concentration for the precipitation depend on the species of the metal chlorides, as well as on pH. At pH 7.4, the critical concentrations are: 3-5 x 10- 5 M for ZnCI2, 5-8 x 10- 5 M for CuCI2, 3 x 10 -4 M for NiCI2 and 1 x 10-3 M for COC12. CaCI2 and MgCI2 are not effective up to about 10-2 M. The different critical concentrations for different cations suggest that precipitation is caused by specific binding of the metal cations to fibrinogen. Interaction of the metal ions with

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curred approximately parallel to the change in turbidity, as shown in Fi#ure 3. Values of the turbidity in Figure 3 represent the stationary values reached about 25-30 minutes after the addition of CuCI 2. However, c,d. spectra were measured several hours after the addition of CuC12, since cell washing and spectra scanning took about one hour. Accordingly, the observed change ofc.d, was caused either by a decrease of the protein concentration due to slow precipitation or by a conformational change accompanying the precipitation. The c.d. spectra also changed on addition of ZnCI 2. However, the concentration range of this change was not always reproducible. Effects of the metal chlorides on the fluorescence of fibrinogen were examined at p H 7.4 and 5.8. The fluorescence intensity was measured at 345 nm when excitation was carried out at 285 nm. Figures 4a and 4b show the ratio of fluorescence intensity of the solutions in the presence (F) and the absence (Fo) ofCuC12 at p H 7.4 and 5.8, respectively. As seen in these figures, the fluorescence of tryptophan residues is quenched effectively by CuC12. However, other metal chlorides did not quench effectively at pH 7.4 or 5.8. F o r example, at p H 5.8, ZnC12, NiCI2, CoC12, and CaCI 2 were ineffective even at concentrations up to 7 x 1 0 - 4 M , 3 . 4 x 1 0 - a M , 1 . 3 x 1 0 - 2 M , and 2.2 x 10- z M, respectively. The effect of turbidity on fluorescence measurements should be significant and was examined in the case of C u C 1 2 a t pH 5.8. In Figure 4b, the absorbance at 285 nm (the excitation wavelength) is also shown. The quenching of fluorescence proceeds steadily with increasing C u C 1 2 concentration without affecting the absorbance up to about 1.5 × 10-5 M. The constant level of the absorbance corresponds to the absorbance due to the protein chromophore. However, a sharp break in fluorescence intensity occurs at the concentration at which turbidity begins to appear. The effect of protein concentration on the quenching of CuC12 was examined at p H 7.4 at two concentrations, 0.0066 and 0.026 g dl- 1. The results are shown in Figure 4a. Values of F/Fo are almost identical but a break occurs at the lower CuCI 2 concentration at high protein concentration.

Interactions of metal ions with buffers The critical concentrations for precipitation depended on the sp.ecies of divalent cations and changed with p H for a _given lOmC species. These results provide useful information on the affinity of metal ions for fibrinogen as well as on the possible binding sites, unless the buffers perturb the metal-protein interactions significantly. Absorption spectra in the visible and ultraviolet regions were taken as a measure of possible interactions between Tris buffer and Cu 2 ÷ ions. Absorption bands appeared with a peak at 240 nm and a shoulder around 270 nm, when CuC12 was mixed with Tris buffer. In Figure 5, the

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absorbance of the solution at 240nm, the absorption maximum, is plotted against the concentration of CuCI2. No absorption bands appeared for other metal chlorides when mixed with Tris buffer. The p H was also measured and the results for Tris buffer are also shown in Figure 5. On addition of CuCI 2, p H decreases considerably which is consistent with the change in the absorption spectra. The results in Figure 5 confirm that the interactions of Tris buffer with metal chlorides are negligible except for CuC12. However, it is generally expected that CuC12 interacts with fibrinogen more strongly than with Tris buffer, at least, at the low buffer concentration used in the present study. Interactions of MES buffer with metal chlorides were examined both by p H measurements and absorption spectra. Little or no change was found up to 4.58 × 10- 4 M for ZnC1 z, 8.05 x 10 - 4 M for CuC12, and 4.64 x 10- 3 M for NiC12. The addition of a small a m o u n t of divalent metal ions should alter the p H much less significantly in this p H range (5.8) than in the neutral region (7.4) even with MES buffer. With this reservation, it is expected that the effects of divalent ions on fibrinogen are not significantly modified by their interactions with MES buffer.

centrations used in their study (0.2 g d1-1) from the present study (0.1 g dl-1 crr lower). Precipitation occurs above certain critical concentrations of metal ions, which depend markedly on the species of ions. Both Ca 2÷ and Mg 2÷ ions do not cause precipitation. F o r the other four species (Co 2÷, Ni 2÷, Cu 2÷, and Zn2+), their order of ability to cause precipitation or gelation is similar in both studies, except for Cu 2÷ ions. However, the abilities were generally lower in the other study than in the present study. The critical concentration for Cu 2+ ions is considerably lower than that for Ni 2 + ions in the present study, while almost the same value is found for both ions in the other study. These different results suggest that the interaction of metal ions with the buffer used m a y have perturbed their interaction with bovine fibrinogen in the Steven et al. study. This is probable because, as shown in the present study, the interaction of Cu 2÷ ions with Tris buffer occurs more strongly than the other three ions and because about a ten times more concentrated buffer is used in the other study. Although Zn 2 ÷ ions are the most effective among the examined species in both studies, the critical concentration is rather different. At p H 7.4, the critical concentration is about 4 x 10- 5 M in the present study, while it is reported to be too low to be detected in the study of Steven et al. 9 As shown in Figure I, the critical concentrations of Zn 2 +, Ni 2 +, and Co 2 + ions become higher by more than one order as the p H changes from 7.4 to 5.8. Since the imidazole group of histidine residues undergoes protonation in the same p H range, the present study suggests that histidine residues are the most probable binding sites for these three ions. For Cu 2 + and Zn z + ions, carboxyl groups are also probable binding sites, as supported by the present data at pH 5.8.

Acknowledgement This work was done with partial financial support from the Ministry of Education, Science and Culture Grant No. 56540256.

References 1 2 3

Discussion Recently, Steven et al. have reported a study on the aggregation of bovine fibrinogen by metal ions 9. They have studied the interaction of fibrinogen with eight kinds of metal ions at p H 7.2, using 0.1 M buffer (either phosphate or Tris). Anions are C I - and SO42- and are not c o m m o n to all metal ions examined. Gelation of fibrinogen occurred on addition of metal ions, while in the present study precipitation occurred. The probable cause of these different findings is different fibrinogen con-

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Marguerie,G. Biochim. Biophys. Acta 1977, 494, 172 Marguerie,G., Chagniel, G. and Suscillon, M. Biochim. Biophys. Acta 1977, 490, 94 Van Ruijven-Vermeer,I. A. M., Nieuwenhuizen, W. and Nooijen, W. J. FEBS Lett. 1978, 93, 177 Nieuwenhuizen,A., Vermond, A., Nooijen, W. J. axadHaverkate, F. FEBS Lett. 1979, 98, 257 Doolittle,R. F. Adv. Protein Chem. 1973, 27, 2 Krantz, S. and Fiedler, H. cited in ref. 5, p. 76 Behlke,J., Krantz, S., Lober, M. and Fiedler, H. cited in ref. 5, p. 76 Fiedler,H., Krantz, S. and Lober, M. cited in ref. 5, p. 76 Steven,F. S., Griffin, M. M., Brown, B. S. and Hulley,T. P. Int. J. Biol. Macromol. 1982, 4, 367 Mihb,lyi, E. Acta Chem. Scandinavica 1950, 4, 351 Edsall,J. T. and Wyman,J. in 'BiophysicalChemistry', 1958,vol. 1, p. 276, Academic Press Inc., New York