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The increase in human antithrombin III tryptophan fluorescence produced by heparin
165
Biochimica et Biophysica Acta, 534 ( 1 9 7 8 ) 1 6 5 - - 1 6 8 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
B B A Report BBA 31251
THE INCREASE IN HUMAN ANTITHROMBIN III TRYPTOPHAN FLUORESCENCE PRODUCED BY HEPARIN
ROLAND EINARSSON*
A B KABI, Research Department, Analytical Chemistry, S-112 8 7 Stockholm (Sweden) ( R e c e i v e d D e c e m b e r 14th, 1977)
Summary The increase in fluorescence of human antithrombin III has been used to study the binding of a semi-synthetic heparin analogue. One molecule of heparin were found to bind with an association constant of 8.9 • 104 M- ' The intrinsic fluorescence of antithrombin III exhibits a fluorescence quant u m yield of 0.17. Upon addition of heparin a marked increase in the protein fluorescence quantum yield is observed.
Human antithrombin III is one of the most important protease inhibitors in blood and is of great importance in the regulation of the haemostatic balance. It is identical with the heparin cofactor and inhibits various plasma coagulation enzymes as well as some enzymes not involved in the coagulation cascade [1--7]. In the presence of heparin the inhibitory activity is increased about 50--100 fold. Heparin causes a structural change in antithrombin III so as to make it a more rapidly acting inhibitor [4, 5, 8, 9]. Previous studies have shown that definite changes in ultraviolet difference and protein t r y p t o p h a n fluorescence occur in connection with the binding of heparin [10, 11]. Further, in preliminary studies it has also been observed that various fractions of heparin appear to produce an increase in fluorescence of varying magnitude. This paper presents an analysis of the fluorescence characteristics of various heparin • antithrombin III complexes and examines the relationship between heparin binding and the fluorescence q u a n t u m yield. Human antithrombin III was prepared by a previously described method [2]. The protein preparation was homogeneous as judged by disc gel electrophoresis and immunoelectrophoresis. Pig mucosal heparin (Vitrum, Sweden) was purified by affinity chromatography on matrixbound anti-
*Present address: Analytical Research, Phaxmacia AB, Box 181, S-751 04 UPpsala, Sweden.
166
thrombin I I I [ 12 ]. Two distinct fractions were obtained, one with high affinity for antithrombin III. The specific activity of the high-affinity fraction was a b o u t 230 B.P. units/mg and the average molecular weight was 11 200, whereas the low-affinity fraction had very little specific activity (about 20 B.P. units/mg) with an average molecular weight of 11 000. The third heparin sample was a semi-synthetic heparin analogue of bovine origin (Luitpold Werk, Munich, G.F.R.). It had a specific activity of a b o u t 90 B.P. units/mg. Fluorescence measurements were performed with an Aminco Bowman SPF equipped with a corrected spectra accessory. In addition, quantum yields were measured by comparing the integrated area under the corrected emission curve of the unknowns with that of a standard [13]. The protein quantum yield of antithrombin III and its complexes with heparin was calculated according to the formula in the Aminco Bowman Operator's Manual. The addition of mucosal heparin to antithrombin III causes an increase in fluorescence intensity and a minor shift to the blue of the emission maximum wavelength [ 1 1 ] . Fluorometric titration of antithrombin III with the semi-synthetic heparin analogue indicated a similar effect even if the enhancement of the protein tryptophan fluorescence was relatively weak. However, this spectral property was followed to measure the binding of the heparin analogue (Fig. 1). Log (Foo--F)/(F--Fo) is plotted against log [H] where Fo, F and Foo are, respectively, the fluorescence intensities of antithrombin III alone, of the protein in the presence of heparin and of antithrombin III saturated with the ligand. [H] is the concentration of heparin analogue. The slope of the curve is 1.1, indicating the formation of a one-to-one complex. The average association constant for the complex equals the value of log [H] at log (F~c--F)/{F--Fo) = 0 [14]. The association constant was found to be 8 . 9 . 104 M - , The addition of low-affinity heparin (mucosal) to antithrombin III did not change the intrinsic tryptophan fluorescence of the protein emission
,og 1.0
0.5
4:o
6.o-,og[HI
-0.5 ~
Fig. 1. S p e c t ~ o f l u o r o m e t r i c d e t e r m i n a t i o n [ 1 4 ] o f t h e a s s o c i a t i o n c o n s t a n t o f a s e m i - s y n t h e t i c h e p a x i n a n a l o g u e to h u m a n a n t i t h r o m b i n III in 0 . 0 5 M Trls b u f f e r , p H 7 . 4 , c o n t a i n i n g 0.2 M g l y c i n e a n d 0 . 0 3 M s o d i u m c h l o r i d e at 2 5 ° C . Fo, F a n d F ~ are t h e relative f l u o r e s c e n c e i n t e n s i t i e s at 3 3 8 n m o f , res p e c t i v e l y , p r o t e i n a l o n e , t h e p r o t e i n in t h e p r e s e n c e o f a c o n c e n t r a t i o n [ H ] o f h e p a r i n a n d o f t h e p r o t e i n s a t u r a t e d w i t h h e p a r i n . T h e t i t r a t i o n d a t a w e r e o b t a i n e d w i t h an e x c i t a t i o n w a v e l e n g t h o f 2 8 5 n m , a n d t h e a n t i t h r o m b i n III c o n c e n t r a t i o n w a s 4.3 ~M.
167 wavelength. Clearly, this heparin fraction with extremely low specific activity is n o t able to induce the necessary structural change in the local environment around the tryptophyl(s) in antithrombin III, and this is used as a sensitive fluorometric indicator. The reason for this might be a very weak interaction, caused by altered chemical structure of the low-affinity heparin molecule, or, alternatively, the binding occurs at a site well separated from the heparin binding region. The latter site does not significantly influence the local environment adjacent to the tryptophyls. The fluorescence quantum yields of antithrombin III, antithrombin III saturated with high-affinity heparin, and antithrombin III saturated with the semi-synthetic heparin analogue, in 0.05 M Tris buffer, containing 0.2 M glycine and 0.03 M sodium chloride at pH 7.4 and at 25°C were determined. The quantum yields were measured by comparison with a reference solution of tryptophan in water at 25°C, assuming that q is equal to 0.13 for tryptophan excited at 280 nm [13]. The quantum yield of antithrombin III was calculated to be 0.17+0.01. This value appears to be relatively high in comparison with other multitryptophan containing proteins [15] and appears to exclude the existence of quenching centres in the proximity of some of the tryptophan fluorophors. The quantum yield of the antithrombin I I I • high-affinity heparin complex is markedly higher (q = 0.24+ 0.01) than that of antithrombin III. The magnitude of the quantum yield suggests a drastic conformational change in the protein molecule [5, 11] which alters the local microenvironment around the tryptophyl(s). Moreover, on the basis of the changes in the fluorometric properties, one may infer the presence of tryptophan residue(s) in the heparin binding region or adjacent to that region. The fluorescence quantum yield of the antithrombin I I I • semi-synthetic heparin complex is markedly lower (q = 0.19+0.01), indicating only a minor conformational change in antithrombin III. The weak binding of this heparin analogue provides further evidence for a limited change in the local environments around the tryptophyls. The central question in the anticoagulant activity of antithrombin III mediated by heparin concerns the very different binding affinity of the various heparin samples. High-affinity heparin (pig mucosa) shows a strong interaction at only one site while gel-filtered mucosal heparin [11] interacts at two sites. The semi-synthetic heparin analogue exhibits a relatively weak interaction on one site. These sites might be partly identical as the binding region for heparin appears to be located in a cleft in which the accessibility of subsites might determine the binding strength [ 1 1 ] . In addition, the heparin binding sites appear to be well separated from the general anion binding site on antithrombin III [ 1 0 ] . The anticoagulant activity of antithrombin III measured in vitro appears to be directly related to the heparin binding strength. The stronger binding site corresponds to the one shown to involve a drastic conformational change in the protein and at the same time high anticoagulant activity. A weak increase in fluorescence indicates a weak interaction, i.e. limited heparin binding at the single heparin binding site and, as a consequence, most of the heparin binding occurs at secondary binding sites. Furthermore, in this case the specific activity of the heparin fraction is markedly lower.
168
Finally, it is interesting to note that although the semi-synthetic heparin analogue exhibits a weak interaction with antithrombin III compared to mucosal heparin and appears to be nearly inactive in vitro, it has a potent effect on antithrombin III in vivo [ 1 6 ] . These observations make synthetic heparin analogues very attractive since they might have properties which selectively mediate the antithrombin III effect in vivo. The author is indebted to Erik Holmer for preparation of the high-affinity and low-affinity heparin fractions. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Abi]dgaard, U. (1968) Scand. J. Clin. Lab. Invest. 21, 89---91 Miller-Anderson, M., Borg, H. and Andersson, L.-O. (1974) Thromb. Res. 5, 439--452 Damus, P.S., Hicks, M. and Rosenberg, R.D. (1973) Nature 2 4 6 , 3 5 5 - - 3 5 7 Rosenberg, R.D. and Damus, P.S. (1973) J. Biol. Chem. 248, 6 4 9 0 - - 6 5 0 5 Rosenberg, R.D. (1975) N. Engl. J. Med. 292, 146--151 Highsmith, R.F. and Rosenberg, R.D. (1974) J. Biol. Chem. 249, 4 3 3 5 - - 4 3 3 8 Burrowes, C.E., Habal, F.M. and Movat, H.Z. (1975) Thromb. Res. 7 , 1 7 8 - - 1 8 3 Bj~rk, I. and Nordenman, B. (1976) Eur. J. Biochem. 68, 507--511 Kurachi, K., Schmer, G., Hermodson, M.A., Teller, D.C. and Davie, E.W. (1976) Biochemistry 15, 368--373 Einarsson, R. (1976) Biochim. Biophys. Acta 4 4 6 , 1 2 4 - - 1 3 3 Einarsson, R. and Andersson, L.-O. (1977) Biochim. Biophys. Acta 490, 104--111 Andersson, L.-O., Barrowcliffe, T.W., Holmer, E., Johnson, E.A. and Sims, G.E.C. (1976) Thromb. Res. 9 , 5 7 5 - - 5 8 3 Chert, R.F., Edelhoch, H. and Steiner, R.F. (1970) Physical Principles and Techniques of Protein Chemistry, Part A, P. 171, Academic Press, New York. Chipman, D.M., Grisaro, V. and Sharon, N. (1967) J. Biol. Chem. 242, 4 3 8 8 - - 4 3 9 4 Teichberg, V.I., Piasse, T., Sorell, S. and Sharon, N. (1972) Biochim. Biophys. Acta 278, 250-257 Thomas, D.P., Michalski, R., Lane, D.A., Johnson, E.A. and Kakkar, V.V. (1977) Lancet i, 120--122
Biochimica et Biophysica Acta, 534 ( 1 9 7 8 ) 1 6 5 - - 1 6 8 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
B B A Report BBA 31251
THE INCREASE IN HUMAN ANTITHROMBIN III TRYPTOPHAN FLUORESCENCE PRODUCED BY HEPARIN
ROLAND EINARSSON*
A B KABI, Research Department, Analytical Chemistry, S-112 8 7 Stockholm (Sweden) ( R e c e i v e d D e c e m b e r 14th, 1977)
Summary The increase in fluorescence of human antithrombin III has been used to study the binding of a semi-synthetic heparin analogue. One molecule of heparin were found to bind with an association constant of 8.9 • 104 M- ' The intrinsic fluorescence of antithrombin III exhibits a fluorescence quant u m yield of 0.17. Upon addition of heparin a marked increase in the protein fluorescence quantum yield is observed.
Human antithrombin III is one of the most important protease inhibitors in blood and is of great importance in the regulation of the haemostatic balance. It is identical with the heparin cofactor and inhibits various plasma coagulation enzymes as well as some enzymes not involved in the coagulation cascade [1--7]. In the presence of heparin the inhibitory activity is increased about 50--100 fold. Heparin causes a structural change in antithrombin III so as to make it a more rapidly acting inhibitor [4, 5, 8, 9]. Previous studies have shown that definite changes in ultraviolet difference and protein t r y p t o p h a n fluorescence occur in connection with the binding of heparin [10, 11]. Further, in preliminary studies it has also been observed that various fractions of heparin appear to produce an increase in fluorescence of varying magnitude. This paper presents an analysis of the fluorescence characteristics of various heparin • antithrombin III complexes and examines the relationship between heparin binding and the fluorescence q u a n t u m yield. Human antithrombin III was prepared by a previously described method [2]. The protein preparation was homogeneous as judged by disc gel electrophoresis and immunoelectrophoresis. Pig mucosal heparin (Vitrum, Sweden) was purified by affinity chromatography on matrixbound anti-
*Present address: Analytical Research, Phaxmacia AB, Box 181, S-751 04 UPpsala, Sweden.
166
thrombin I I I [ 12 ]. Two distinct fractions were obtained, one with high affinity for antithrombin III. The specific activity of the high-affinity fraction was a b o u t 230 B.P. units/mg and the average molecular weight was 11 200, whereas the low-affinity fraction had very little specific activity (about 20 B.P. units/mg) with an average molecular weight of 11 000. The third heparin sample was a semi-synthetic heparin analogue of bovine origin (Luitpold Werk, Munich, G.F.R.). It had a specific activity of a b o u t 90 B.P. units/mg. Fluorescence measurements were performed with an Aminco Bowman SPF equipped with a corrected spectra accessory. In addition, quantum yields were measured by comparing the integrated area under the corrected emission curve of the unknowns with that of a standard [13]. The protein quantum yield of antithrombin III and its complexes with heparin was calculated according to the formula in the Aminco Bowman Operator's Manual. The addition of mucosal heparin to antithrombin III causes an increase in fluorescence intensity and a minor shift to the blue of the emission maximum wavelength [ 1 1 ] . Fluorometric titration of antithrombin III with the semi-synthetic heparin analogue indicated a similar effect even if the enhancement of the protein tryptophan fluorescence was relatively weak. However, this spectral property was followed to measure the binding of the heparin analogue (Fig. 1). Log (Foo--F)/(F--Fo) is plotted against log [H] where Fo, F and Foo are, respectively, the fluorescence intensities of antithrombin III alone, of the protein in the presence of heparin and of antithrombin III saturated with the ligand. [H] is the concentration of heparin analogue. The slope of the curve is 1.1, indicating the formation of a one-to-one complex. The average association constant for the complex equals the value of log [H] at log (F~c--F)/{F--Fo) = 0 [14]. The association constant was found to be 8 . 9 . 104 M - , The addition of low-affinity heparin (mucosal) to antithrombin III did not change the intrinsic tryptophan fluorescence of the protein emission
,og 1.0
0.5
4:o
6.o-,og[HI
-0.5 ~
Fig. 1. S p e c t ~ o f l u o r o m e t r i c d e t e r m i n a t i o n [ 1 4 ] o f t h e a s s o c i a t i o n c o n s t a n t o f a s e m i - s y n t h e t i c h e p a x i n a n a l o g u e to h u m a n a n t i t h r o m b i n III in 0 . 0 5 M Trls b u f f e r , p H 7 . 4 , c o n t a i n i n g 0.2 M g l y c i n e a n d 0 . 0 3 M s o d i u m c h l o r i d e at 2 5 ° C . Fo, F a n d F ~ are t h e relative f l u o r e s c e n c e i n t e n s i t i e s at 3 3 8 n m o f , res p e c t i v e l y , p r o t e i n a l o n e , t h e p r o t e i n in t h e p r e s e n c e o f a c o n c e n t r a t i o n [ H ] o f h e p a r i n a n d o f t h e p r o t e i n s a t u r a t e d w i t h h e p a r i n . T h e t i t r a t i o n d a t a w e r e o b t a i n e d w i t h an e x c i t a t i o n w a v e l e n g t h o f 2 8 5 n m , a n d t h e a n t i t h r o m b i n III c o n c e n t r a t i o n w a s 4.3 ~M.
167 wavelength. Clearly, this heparin fraction with extremely low specific activity is n o t able to induce the necessary structural change in the local environment around the tryptophyl(s) in antithrombin III, and this is used as a sensitive fluorometric indicator. The reason for this might be a very weak interaction, caused by altered chemical structure of the low-affinity heparin molecule, or, alternatively, the binding occurs at a site well separated from the heparin binding region. The latter site does not significantly influence the local environment adjacent to the tryptophyls. The fluorescence quantum yields of antithrombin III, antithrombin III saturated with high-affinity heparin, and antithrombin III saturated with the semi-synthetic heparin analogue, in 0.05 M Tris buffer, containing 0.2 M glycine and 0.03 M sodium chloride at pH 7.4 and at 25°C were determined. The quantum yields were measured by comparison with a reference solution of tryptophan in water at 25°C, assuming that q is equal to 0.13 for tryptophan excited at 280 nm [13]. The quantum yield of antithrombin III was calculated to be 0.17+0.01. This value appears to be relatively high in comparison with other multitryptophan containing proteins [15] and appears to exclude the existence of quenching centres in the proximity of some of the tryptophan fluorophors. The quantum yield of the antithrombin I I I • high-affinity heparin complex is markedly higher (q = 0.24+ 0.01) than that of antithrombin III. The magnitude of the quantum yield suggests a drastic conformational change in the protein molecule [5, 11] which alters the local microenvironment around the tryptophyl(s). Moreover, on the basis of the changes in the fluorometric properties, one may infer the presence of tryptophan residue(s) in the heparin binding region or adjacent to that region. The fluorescence quantum yield of the antithrombin I I I • semi-synthetic heparin complex is markedly lower (q = 0.19+0.01), indicating only a minor conformational change in antithrombin III. The weak binding of this heparin analogue provides further evidence for a limited change in the local environments around the tryptophyls. The central question in the anticoagulant activity of antithrombin III mediated by heparin concerns the very different binding affinity of the various heparin samples. High-affinity heparin (pig mucosa) shows a strong interaction at only one site while gel-filtered mucosal heparin [11] interacts at two sites. The semi-synthetic heparin analogue exhibits a relatively weak interaction on one site. These sites might be partly identical as the binding region for heparin appears to be located in a cleft in which the accessibility of subsites might determine the binding strength [ 1 1 ] . In addition, the heparin binding sites appear to be well separated from the general anion binding site on antithrombin III [ 1 0 ] . The anticoagulant activity of antithrombin III measured in vitro appears to be directly related to the heparin binding strength. The stronger binding site corresponds to the one shown to involve a drastic conformational change in the protein and at the same time high anticoagulant activity. A weak increase in fluorescence indicates a weak interaction, i.e. limited heparin binding at the single heparin binding site and, as a consequence, most of the heparin binding occurs at secondary binding sites. Furthermore, in this case the specific activity of the heparin fraction is markedly lower.
168
Finally, it is interesting to note that although the semi-synthetic heparin analogue exhibits a weak interaction with antithrombin III compared to mucosal heparin and appears to be nearly inactive in vitro, it has a potent effect on antithrombin III in vivo [ 1 6 ] . These observations make synthetic heparin analogues very attractive since they might have properties which selectively mediate the antithrombin III effect in vivo. The author is indebted to Erik Holmer for preparation of the high-affinity and low-affinity heparin fractions. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Abi]dgaard, U. (1968) Scand. J. Clin. Lab. Invest. 21, 89---91 Miller-Anderson, M., Borg, H. and Andersson, L.-O. (1974) Thromb. Res. 5, 439--452 Damus, P.S., Hicks, M. and Rosenberg, R.D. (1973) Nature 2 4 6 , 3 5 5 - - 3 5 7 Rosenberg, R.D. and Damus, P.S. (1973) J. Biol. Chem. 248, 6 4 9 0 - - 6 5 0 5 Rosenberg, R.D. (1975) N. Engl. J. Med. 292, 146--151 Highsmith, R.F. and Rosenberg, R.D. (1974) J. Biol. Chem. 249, 4 3 3 5 - - 4 3 3 8 Burrowes, C.E., Habal, F.M. and Movat, H.Z. (1975) Thromb. Res. 7 , 1 7 8 - - 1 8 3 Bj~rk, I. and Nordenman, B. (1976) Eur. J. Biochem. 68, 507--511 Kurachi, K., Schmer, G., Hermodson, M.A., Teller, D.C. and Davie, E.W. (1976) Biochemistry 15, 368--373 Einarsson, R. (1976) Biochim. Biophys. Acta 4 4 6 , 1 2 4 - - 1 3 3 Einarsson, R. and Andersson, L.-O. (1977) Biochim. Biophys. Acta 490, 104--111 Andersson, L.-O., Barrowcliffe, T.W., Holmer, E., Johnson, E.A. and Sims, G.E.C. (1976) Thromb. Res. 9 , 5 7 5 - - 5 8 3 Chert, R.F., Edelhoch, H. and Steiner, R.F. (1970) Physical Principles and Techniques of Protein Chemistry, Part A, P. 171, Academic Press, New York. Chipman, D.M., Grisaro, V. and Sharon, N. (1967) J. Biol. Chem. 242, 4 3 8 8 - - 4 3 9 4 Teichberg, V.I., Piasse, T., Sorell, S. and Sharon, N. (1972) Biochim. Biophys. Acta 278, 250-257 Thomas, D.P., Michalski, R., Lane, D.A., Johnson, E.A. and Kakkar, V.V. (1977) Lancet i, 120--122