Interaction of rabbit hemopexin with bilirubin

57

Biochimica et Btophysica Acta, 5 3 2 ( 1 9 7 8 ) 5 7 - - 6 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 3 7 8 2 4

INTERACTION OF RABBIT HEMOPEXIN WITH BILIRUBIN

W I L L I A M T. M O R G A N a, U R S U L A M U L L E R - E B E R H A R D a a n d A N G E L O A L A M O L A b

a Department o f Biochemistry, Scripps Chnic and Research Foundation, La Jolla, Calif 92037 and b Bell Laboratories, Murray Hill, N.J. 07974 (U.S A ) (Received July 1 8 t h , 1 9 7 7 )

Summary The interaction of hemopexin with bdirubin was characterized by spectrophotometric, fluorimetric and circular dmhroic techniques. Hemopexin rapidly forms an equimolar complex with libirubin that has an apparent dissociation constant Kd, of 7.5 • 10 -7 M. The association alters the absorption band of bilirubin near 450 nm, quenches the fluorescence of tryptophan residues of hemopexin, enhances the fluorescence of bdirubin, and mduces strong ellipticity extrema in bilirubin of --60 • 103 deg • cm 2 • dmol -~ at 465 nm and +70 • 10 ~ deg • cm 2 • dmo1-1 at 415 nm. However, the conformation-sensitive ellipticity band at 231 nm of hemopexin is not altered. In displacement experiments using circular dichroism, heme readily replaced bound bllirubin, indicating that bilirubin and heme are bound at the same site on hemopexin. Even at molar rahos of hemopexin to albumin of 3 to 1, human serum albumin removes bilirubin from hemopexin. Hemopexin is thus unlikely to have a role in the transport of bihrubin in serum.

Introduction The interaction of hemopexin with heme, forming a low spin, equimolar complex [1--3] and with naturally occurring and synthetic porphyrins [4--7] has been extensively studied. This reformation is sought for two reasons: first, to increase understanding of the biological function of hemopexin in the transport of heme and porphyrins; and second, to define the characteristics of its single heme-binding site. Current evidence indicates that the binding site of hemopexin will accommodate porphyrins containing a wide variety of peripheral substituents, includmg mesotetraphenyl porphyrins [7]. A major determinant of the binding affinity Abbrewahon: Me2 SO, dlmethvl sulfoxlde.

58 appears to be the presence of iron in the porphyrin with which two h~stidine residues of hemopexin coordinate. For example, hemopexin binds heme with a dissociation constant (Kd) near 10 -~3 M [8] and protoporphyrin with a Kd near 10 -6 M [ 5]. The large contribution of bis-histidyl coordination is supported by the lowered affinity for heme exhibited by bromoacetate-treated hemopexln, K d near 10 -6 M [9]. While studymg serum proteins which participate in organic anion transport, we detected the binding of bilirubin by hemopexin using fluorescence techniques. This finding was of interest since bilirubin can exert toxic effects, especially in predisposed neonates [10]. To determine whether hemopexin has a role in the physiological transport of bilirubin and to further characterize the heme-binding site of hemopexin, we have studied the interaction of hemopexin with bilir~abin. Since the interaction of h u m a n serum albumin with bilirubin is well-described [11--12] and has physiological importance [13], we directly compared albumin and hemopexin in several experiments. In this report, the results of studies on the interaction of hemopexin with bilirubin employing spectrophotometric, fluorimetric and circular dichroic techniques are presented. Hemopexin thus joins several other proteins reported to bind bilirubin, such as serum albumin [11,12], hgandin [14], aminoazodyebinding protein A [15] myelin basic protein [16], and apo-myoglobin [17], and may be the previously unidentified H-region serum protein reported to bind bilirubin [ 18]. Materials and Methods Hemopexin was purified from rabbit and human serum and the purity of the preparations assessed as previously described [1]. Human serum albumin, obtained from Kabi AB (lot No. 30299) and Behringwerke AG (lot No. 2545), was passed through a 2.5 × 80 cm column of AcA44 (LKB) to remove dimers. Concentrations of h e m o p e x m and albumin were measured using millimolar extinction coefficmnts at 280 nm of 110 [19] and 36 [20], respectively. Solutions of heme (Eastman, lot No. 691) and bilirubin IX (Eastman or Sigma) were prepared fresh in dimethylsulfoxide (Me2SO), and carefully protected from light. Concentrations were determined using millimolar extinction coefficients in Me2SO of 170 at 404 nm for heme [21] and 64 at 455 nm [22] for bilirubin. Mixtures of protein with ligand were prepared by adding a small volume of ligand in Me2SO to protein in phosphate-buffered saline or 0.1 M sodium phosphate buffer, pH 7.1 and gently stirring. Final dilutions and concentrations of Me2SO were less than 5% in all experiments. Spectrophotometric measurements were recorded with a Cary 118C instrument at 22°C. Fluorescence measurements were obtained using a Perkin-Elmer MPF-44A spectrofiuorimeter at 22°C [6], and fluorescence quenching data treated by the method of Lehrer and Fasman [23]. The absorbance at 280 nm was maintained below 0.08 and all values were corrected for dilution and screening effects by corrections based on ovalbumin-bilirubin control titrations. FIFo is the relative fluorescence defined as the observed fluorescence, F, divided by the initial fluorescence, F0. Circular dichrolsm (CD) spectra were recorded on a Cary 61 instrument at

59 25°C using 1 cm cuvets [4]. CD results, expressed in units of degree • cm 2 • dmol-', were calculated on the basis of protein or ligand concentratmn; the basis of calculation is presented in the text for each experiment. Results and Discussion The absorption spectra of bilirubin-hemopexin, bilirubin-albumin and bilirubin alone from 550 to 350 nm are shown in Fig. 1. The same concentration of bilirubin was present in each sample (0.3 pM), and the molar ratio of bilirubin to protein was 1 to 5 to minimize free bilirubin. The spectrum of the bilirubinhemopexin sample 1 min after mixing indicates that a complex is rapidly formed. Unlike the red-shift seen with bilirubin-albumin, a slight blue-shift m observed with hemopexin-bilirubin. Alternatively, one could argue that there are no spectral shifts but rather a change in the ratio of intensities of two absorption bands, perhaps associated with two different conformers of bihrubin. It is interesting that the hemopexin-bilirubin spectrum resembles that of bflirubin in human serum from which albumin was removed (Lamola, A., unpublished observation). When bilirubin was mixed with heme-hemopexin, there was no change m the difference absorption spectrum; however, when heme was mixed with bilirubinhemopexin, there was a rapid change to the normal heme-hemopexin difference spectrum (not shown). Incubation of the samples in the dark at ambient temperature for 20 min produced a noticeable general decrease in absorption of the spectrum of bilirubin-hemopexin, and smaller decreases in the spectra of the other samples. This change in the bilirubin chromophore probably results from its known lability, even in the dark (e.g. ref. 22). Addition of bilirubin to hemopexin produced an immediate decrease in the fluorescence of tryptophan residues (Fig. 2), similar to the quenching of hemo-

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Fig. 1. A b s o r p t i o n spectra o f blhrubin, b f l ~ u b m - h e m o p e x i n and bilirubm-albumm. S p e c t r a were recorded wltlu n 3 min o f a d d i n g bilirubm (0.3 pM) t o r a b b i t h e m o p e x m (1.5 pM) o r t o h u m a n serum a l bumi n (1.5 pM) at 22°C in 0.1 M s o d i u m p h o s p h a t e buffer, pH 7.15.

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Fig. 2. Q u e n c h i n g o f h e m o p e x m f l u o r e s c e n c e b y b i h r u b m . T h e e x c i t a t i o n w a s at 2 8 0 n m . H e m o p e x i n , 1 # M . w a s m p h o s p h a t e - b u f f e r e d saline, p H 7 . 4 . T h e s p e c t r u m w a s r e - r e c o r d e d a f t e r a d d i n g 1 a n d 2 equival e n t s o f bHLrUbm, w i t h d f l u t m n less t h a n 5%. Fig. 3. E n h a n c e m e n t o f b i h r u b i n f l u o r e s c e n c e b y h e m o p e x i n . T h e e x m t a t i o n w a s at 4 3 0 rim. H e m o p e x m , 1 p M , w a s a d d e d to 1 ~ M b f l i r u b i n m p h o s p h a t e - b u f f e r e d s a h n e , a n d t h e s p e c t r u m r e - r e c o r d e d .

pexin produced by heme and other porphyrins [5,6,7]. The increase in fluorescence of bilirubin when bound to protein [11] was also observed with hemopexin (Fig. 3). Addition of bilirubin to heme-hemopexin produced no further quenching of hemopexin's fluorescence and no enhancement of the fluorescence of bilirubin. Like the optical spectra, the fluorescence of hemopexin-bilirubin complexes also changed with time. For this reason, all titration experiments were completed within 20 min. As shown in Fig. 4, the quenching of fluorescence increased with increasing concentrations of bdlrubin. These measurements were used to estimate the stoichiometry and affinity of the interactions between bilirubin and rabbit hemopexin (Fig. 5). The indicated stoichiometry of binding is 1, whmh is supported by the results of titrations per-

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Fig. 4. Q u e n c h i n g o f h e m o p e x l n a n d a l b u m i n f l u o r e s c e n c e b y b i h r u b m . F l u o r e s c e n c e m t e n s i t m s w e r e m e a s u r e d at 2 2 O c a f t e r a d d i t i o n s o f b l h r u b m . T h e e x c i t a t i o n w a s at 2 8 0 n m a n d e m i s s i o n w a s m o n i t o r e d at 3 4 5 n m . T h e c o n c e n t r a t i o n s o f p r o t e i n w e r e . 0 . 9 p M for h e m o p e x i n , a n d 2 p M f o r h u m a n s e r u m album i n In 0 . 1 M s o d i u m p h o s p h a t e , p H 7 . 1 5 . Fig. 5. D o u b l e l o g t r e a t m e n t o f b i l i r u b i n - h e m o p e x m b i n d i n g t i t r a t i o n . C o n d i t i o n s are t h e s a m e as in Fig. 4. T h e d a t a w e r e t r e a t e d b y t h e m e t h o d o f L e h r e r a n d F a s m a n [ 2 3 ]

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F i g 6. C i r c u l a r d l c h r o l s m s p e c t r a o f b l h r u b m - , h e i n e - , a n d b f l t r u b m - h e m e - a l b u m m . C D s p e c t r a o f t h e s e e q u l m o l a r m i x t u r e s were r e c o r d e d at 2 5 ° C m 0.1 M sochum p h o s p h a t e b u f f e r w i t h i n a few m i n u t e s of adding hgand to human serum albumin. The spectra were recorded again after additmn of the second h g a n d . T h e m o l a r e l h p t m l t m s w e r e c a l c u l a t e d o n t h e b a s i s o f b l h r u b m , 1 . 5 • 1 0 -5 M, o r h e m e , 1.5 • 1 0 -5 M , a n d a l b u m i n f o r t e r n a r y c o m p l e x e s , 1.5 1 0 -5 M. F i g . 7. C x r c u l a r d i c h r o l s m s p e c t r a o f b l h r u b m - , h e i n e - a n d b i h m b m - h e m e - h e m o p e x m . Conditions were the s a m e a s l n F i g 6. T h e m o l a r e l h p t m l t l e s w e r e c a l c u l a t e d o n t h e b a s i s o f b t h r u b m , 1 . 5 - 1 0 - 5 M , h e i n e , 1 . 5 1 0 -5 M, a n d h e m o p e x m f o r t e r n a r y c o m p l e x e s , 1 . 5 - 1 0 -5 M.

formed at higher concentrations of protein (not shown) and of competition experiments with heme (see below). The apparent Kd is approximately 7.5 • 10 -7 M, assuming a 1 : 1 complex of bilirubin with hemopexin. This value is considerably higher, i.e. lower affinity, than the K d values reported for human serum albumin-bilirubin complexes (10 -s M to 10 -9 M) [11,12], and is of the same order of magnitude as ligandin, protein A, and apo-myoglobin-bihrubin complexes [14,15,17]. More recent results (Lamola, Blumberg and Elsinger, m preparation) indicate that the Kd of defatted human serum albumin-bilirubin is 1.4 • 10 -7 M. Circular dichroism has proven useful in characterizing the interaction of blhrubin with albumin, ligandin and aminoazodye-binding protein A [11,14,15]. Two types of CD spectra have been observed for protein-bound bilirubin; one, e.g. bilirubin-human serum albumin at alkaline pH (Fig. 6), displays positive elliptmity at longer wavelength and negative elliptlcity at shorter wavelength; the other more c o m m o n type is the inverse of this [14,15,24]. Hemopexin has a spectrum of the latter type {Fig. 7). These characteristic CD properties of protein-bound bilirubin are thought to arise from the chlral properties of the twisted bllirubin pyrroles [25], also observed in the crystal structure [26]. This structure is apparently induced in the chromophore by its interaction with the proteins that bind it since the optical activity of bilirubin in solution is negligible. Bihrubin-hemopexin has ellipticity extrema of --60 • 103 deg • cm 2 • dmol -I at 465 nm and 70 • 103 deg • cm 2 • dmo1-1 at 415 nm (Fig. 7). The spectra were observed immediately after mixing, indicating rapid formation of the complex. Like the absorption and fluorescence spectra, the bilirubin-hemopexin CD spectrum decayed measurably after several minutes. Similar significant breakdown of bilirubin was found in control experiments in which bilirubin-albumin, bilirubin-hemopexin or bilirubin alone were incubated in the dark at room tem-

62 perature for 20 h (not shown). Unlike heme, bilirubin bound to hemopexin did not alter the conformationally sensitive ellipticity of hemopexin at 231 nm (not shown) [4]. Titration of hemopexin with bilirubin using CD techniques, carried out on separate samples of protein to minimize time-dependent changes, showed an apparent equimolar stoichiometry of binding (not shown). Displacement experiments were performed to confirm that bihrubin and heme are bound at the heme-binding site of hemopexin in a mutually exclusive fashion (Fig. 7). The heme-hemopexin complex has a positive ellipticity of 70 • 103 deg. cm 2 • dmo1-1 at 418 nm due to induced extrinsic ellipticity in heme when bound; unbound heme has no such signal. When one equivalent of heme was mixed with hemopexin immediately after one equivalent of bilirubin, the typical bilirubin-hemopexin spectrum was replaced within 3 min by a characteristic heme-hemopexin spectrum. This spectrum did not change over 20 h. When bilirubin was added after heme, the heme-hemopexin CD spectrum, hke the absorbance or fluorescence spectra (not shown), was not affected (Fig. 7). This indicates that bilirubin dissociates readily from hemopexin allowing the much more tightly bound heme access to the binding site of hemopexin. Another series of experiments was conducted to directly test the apparent differences in affimty for bilirubin displayed by hemopexin and albumin. As shown in Fig. 8, the CD spectrum of bllirubin-hemopexin was rapidly altered after albumin was added to a near-characteristm bilirubin-albumin spectrum. This was observed even at molar ratios of hemopexin : albumin : bilirubin of 3 : 1 : 1. Conversely, addition of hemopexin to bilirubin-albumin had little effect (Fig. 8). Experiments in which hemopexin : albumin : bihrubm molar ratios were 1 : 1 : 2 (not shown) were equivocal as to whether bilirubin-hemopexin association took place. This has not been resolved because of technical difficulties in distinguishing bilirubin bound to secondary sites of albumin in a complex mixture. Heme bound to albumin produces a characteristic CD spectrum in the Soret region (Fig. 6). The primary binding sites of albumin for heme and bihrubin are different [20,27]. Consequently, the CD signals of heme and bllirubin bound to albumin (Fig. 6) do not depend strongly on the order of addition of ligand. A series of expemments was carried out on equlmolar mixtures of albumin, heme, bilirubin and hemopexin. As shown in Fig. 9, after formation of the heme-bilirubin-albumin complex, the CD spectrum immediately after addition of hemopexin is largely unchanged. However, there is a slight change in the negative elliptmity near 415 nm whmh can be attributed to formation of hemehemopexin with its positive ellipticity in that area. After 20 h, the spectrum shows a distinct peak near 418 nm attributable to heme-hemopexin. The spectrum of bilirubin-albumin is still evident, but it is largely reduced. These results suggest that some heme is loosely bound to albumin and immediately available for binding by hemopexin. After incubation, nearly all the heme present has been bound by hemopexin. This is consistent with the results of recent work on the transfer of heme from albumin to hemopexin [28] in which it was shown that heme bound at the tight site of albumin was transferred to hemopexin over several hours, whereas heme bound at secondary sites was more readily transferred. In conclusion, the present results indmate that hemopexm binds bilirubin,

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F i g . 8. E f f e c t s o f a d d e d p r o t e i n o n t h e c i r c u l a r d i c h r o i s m o f b ] h r u b m - p r o t c m c o m p l e x e s . C o n d i t i o n s w e r e t h e s a m e as m F i g . 6. A f t e r r e c o r d i n g t h e s p e c t r u m o f t h e fncst l i g a n d - p r o t e m c o m p l e x , o n e e q u i v a l e n t of the s e c o n d p r o t e i n was a d d e d a n d the s e c o n d s p e c t r u m r e c o r d e d w i t h i n a few m i n u t e s . The m o l a r elhpticlties were calculated on the basis of bfllrubm concentration 1 . 8 • 1 0 -5 M m t h e f i r s t s c a n a n d 1 . 5 1 0 -5 M a f t e r a d d i t i o n o f s e c o n d p r o t e i n . F i g . 9. E f f e c t s o f a d d e d h e m o p e x m o n t h e c i r c u l a r d m h r o i s m o f b t h r u b l n - h e m e - a l b u m m . C D s p e c t r a w e r e r e c o r d e d u n d e r t h e s a m e c o n d i t i o n s as d e s c r i b e d m F i g s . 6 a n d 8 w i t h i n a f e w m i n u t e s o f a d d i t i o n o f hgand or hemopexm and after 20 h m the dark at room temperature. The molar elhptlclties of these equlm o l a r m i x t u r e s w e r e c a l c u l a t e d o n t h e b a s i s o f t h e i n i t i a l a l b u m i n c o n c e n t r a h o n , 1.5 • 1 0 -5 M, a n d t h e albumin eonecntrahon m subsequent scans corrected for dilution by added hgand or hemopexm.

apparently at its heme-bindmg site, forming an equimolar complex. This interaction is of possible physiologmal significance only at extremely high bilirubin levels, since albumin not only has a greater affinity for bilirubin but also occurs in the circulation at a b o u t 40 times higher concentration. Recent results (Lamola, Blumberg and Eisinger, in preparation) directly demonstrate that in human blood bilirubin first saturates albumin (at one blhrubin per albumin), then excess bilirubin becomes associated with the erythrocyte membrane (approximately 2) and with other proteins (~). After these serum proteins, which 3 have a small capacity compared to the albumin normally present, are saturated, additional bilirubin associates with the erythrocyte membrane. In pathophysiological conditions where both high bilirubin and hemolysis exist, the possible ability of hemopexin to aid m preventing kermcterus would be dimimshed since heme would abolish the hemopexin-bilirubln interaction and deplete hemopexin [7]. It is interesting that bllirubin, a non-planar molecule both in its proteinbound form [25] and in crystals [26], can interact with hemopexin. Previous work has demonstrated that hemopexin can bind planar porphyrins bearing a wide variety of peripheral substituents [7] and that heme bound to hemopexin seems relatively exposed to solvent [29]. The present results therefore support the concept that the heme-binding site of hemopexin is sterically non-restrictive. Acknowledgements This work was supported by grants from the National Institutes of Health (HD-09252 and AM-16737). William T. Morgan is a recipient of a Research

64

Career Development Award from the National Institutes of Health (AM00110). The expert technical assistance of Mr. Roger P. Sutor is gratefully acknowledged. References 1 Hrkal, Z. and Muller-Eberhard, U. (1971) Btoehemlstry 10, 1746--1750 2 Bearden, A.J., Morgan, W.T. and Muller-Eberhard, U. (1974) Biochem. Blophys. Res C ommun 61, 265--272 3 Alsen, P., Letbman, A., H a m s , D.C. and Moss, T. (1974) J. Biol. Chem. 249, 6 8 2 4 - - 6 8 2 7 4 Morgan, W.T. and Muller-Eberhard, U. (1972) J. Biol. Chem. 247, 7181--7187 5 Seer:c, V.L. and Muller-Eberhard, U. (1973) J. Biol. Chem. 248, 3 7 9 6 - - 3 8 0 0 6 Morgan, W.T., Sutor, R.P., Muller-Eberhard, U. and Koskelo, P. (1975) BiochLrn. Blophys. Acta 400, 415--422 7 Morgan, W.T. (1976) Annals Clin. Res. 8, Suppl. 17, 223--232 8 Hrkal, Z., V o d r ~ k a , Z. and Kalousek, I. (1974) Eur. J. Blochem. 43, 73--78 9 Morgan, W.T. and Muller-Eberhard, U. (1976) Arch. Bxochem. Biophys. 1 7 6 , 4 3 1 - - 4 4 1 10 Powell, L.W. (1972) Seminars Hematol. 9, 91--105 11 Beaven, G.H., d'Albls, A. and Gratzer, W.B. (1973) Eur. J. Blochem. 3 3 , 5 0 0 - - 5 1 0 12 Jacobsen, J. (1969) FEBS Lett. 5, 112--114 13 Thaler, M.M. (1972) Seminars Hematol. 9, 107--112 14 Kamtsaka, K., Llstowsky, I., Gatmaitan, Z. and Arias, I.M. (1975) Biochemtstrv 14, 2175--2180 15 Tipping, E., Ketterer, B., Christodoulides, L. and Enderby, G. (1976) Bmchem. J. 1 5 7 : 2 1 1 - - 2 1 6 16 Gurba, P.E. and Zand, R. (1974) Blochem. Biophys. Res. Commun. 58, 1142--1147 17 Lind, K.E. and M~bller, J.V. (1976) Biochem. J. 155, 669--678 18 AthanasstadLs, S., Chopra, D.R., Ftsher, M.A. and McKenna, J. (1974) J. Lab. Chn. Med. 83, 968-976 19 Seery, V.L., Hathaway, G. and Muller-Eberhard, U. (1972) Arch. Blochem. Blophys. 150, 269--272 20 Beaven, G.H., Chen, S.H., d'AIbls, A. and Gratzer, W.B. (1974) Eur. J. Blochem. 41, 539--546 21 Brown, S.B. and Lantzke, I.R. (1969) Blochem. J. 1 1 5 , 2 7 9 - - 2 8 5 22 Llghtner, D.A., Cu, A., McDonagh, A.F. and Palma, L.A. (1976) Blochem. Blophys. Res. Commun. 69, 648--657 23 Lehrer, S.S. and Fasman, G.D. (1966) Btochem. Blophys. Res. Commun. 23, 133--138 24 Blauer, G. Lavie, E. and Sflfen, J. (1977) Blochlm. Btophys. Acta 492, 64--69 25 Blauer, G. and Wagnldre, G. (1975) J. Am. Chem. Soc. 97, 1949--1954 26 Bonnett, R., Davies, J.E and Hursthouse, M.B. (1976) Nature 262, 326--328 27 Lmm, H.H. and Muller-Eberhard, U. (1971) Blochem. Blophys. Res. Commun. 4 2 , 6 3 4 - - 6 3 9 28 Morgan, W.T., Lmm, H.H., Sutor, R.P and Muller-Eberhard, U. (1976) Btochim. Btophys. Acta 444, 435--445 29 Morgan, W.T., Sutor, R.P. and Muller-Eberhard, U. (1976) Btochtm. Biophys. Acta 434, 311--323