Lanthanide ion probes of structure in biology

135

Biochimica et Biophysica Acta, 578 (1979) 135--144 © Elsevier/North-Holland Biomedical Press

BBA 38174

L A N T H A N I D E ION PROBES OF S T R U C T U R E IN BIOLOGY E N V I R O N M E N T A L L Y SENSITIVE FINE S T R U C T U R E IN LASER-INDUCED TERBIUM(III) LUMINESCENCE

DANIEL R. SUDNICK and WILLIAM DEW. HORROCKS, Jr.

Department of Chemistry, The Pennsylvania State University, University Park, PA 16802 (U.S.A.) ( R e c e i v e d November 28th, 1978)

Key words: Luminescence; Ln3+ probe; Tb(III), (Laser induced)

Summary The 488 nm line of the CW argon ion laser provides a convenient visible source for the direct excitation of the emissive SD4 state of the Tb(III) ion. R o o m temperature emission spectra of Tb(III) in a variety of environments have been examined under relatively high resolution. The samples studied include structurally well-characterized crystalline solids, model chelate complexes in solution and Tb(III) b o u n d to the enzyme thermolysin and the protein parvalbumin. The fine structure in the emissions is caused by ligand field splittings of both ground and excited state J manifolds. These spectra provide signatures sensitive to the immediate coordination environment of the Tb(III) ion. Solid state/solution state structural comparisons are made. The emission fine structure reveal differences between the EF side calcium-binding sites of parvalbumin and the calcium site 1 of thermolysin.

Introduction The utility of trivalent lanthanide ions, Ln(III), as replacement probes for calcium b o u n d to proteins has been discussed in several recent reviews [1--4]. Our research in this area has concentrated on the development and exploitation of lanthanide ion luminescence to probe the structural details of metalbinding sites. We have found that it is possible to overcome the inherent weakness of Ln(III) ion luminescence by directly exciting Ln(III) ion levels using Abbreviations: CPL, circularly polarized luminescence; DPA, dipicoUnic acid; continuous wave.

136

the intense visible radiation from pulsed dye lasers. We have shown [5--7] how the measurement of the luminescence emission lifetimes of Tb(III) and Eu(III) in both H20 and 2H20 solutions provides an accurate measure of the number of water molecules coordinated to the Ln(III) ion when the ion is b o u n d to a macromolecule. The present paper explores the biological probe potential of another aspect of Ln(III) ion luminescence, namely the fine structure revealed by examination of the emissions under relatively high spectral resolution. The 488 nm line of a CW argon ion laser provides a convenient visible source for the direct excitation of the SD4 Tb(III) ion emissive level. This paper will be concerned with Tb(III) ions only. With the exception of our own work dealing with luminescence lifetime measurements [5--7] or interionic energy transfer [8], most of the biologically oriented research involving Tb(III) luminescence has been concerned with the stoichiometry and strength of Tb(III) ion binding. In all such cases the sensitization of Tb(III) luminescence, which frequently occurs u p o n binding of this ion to a protein (protein aromatic amino acid to Tb(III) energy transfer), has been a necessary requirement. The only research which has concerned itself with the study of emission spectra under high resolution is that of Gafni and Steinberg [9] who observed identical emission spectra for Tb(III) bound to transferrin and to conalbumin. In addition, these authors measured the circularly polarized luminescence (CPL) of the proteins. Recently Brittain et al. [ 10] did similar CPL experiments on approximately 40 other proteins. Many of these proteins exhibited no CPL and those that did produced one or the other of only two CPL patterns. It is our purpose here to survey the sensitivity of Tb(III) emissions to changes in the coordination environment and to demonstrate the feasibility of using a continuous wave visible laser to excite protein-bound Tb(III) ions at low concentration in solution with a signal to noise ratio high enough for the satisfactory resolution of fine structure. To this end we have examined a variety of structurally well-characterized crystalline solids and a series of model chelate complexes in solution. We have also performed experiments on Tb(III) b o u n d to the calcium-binding protein, parvalbumin, from carp muscle and to the thermostable endoproteinase, thermolysin. Experimental Instrumentation. The luminescence emission spectra were obtained using a Spex 'Ramalog' Raman spectrometer (Spex Industries, Metuchen, NJ). The SD4 excited state manifold of Tb(III) was excited by the 488 nm line of a Spectra Physics Model 164-3 argon ion laser. A minimal pump power of 250--500 mW proved sufficient to obtain excellent spectra. The emitted radiation was focused onto the slits of a Spex Model 1401 double m o n o c h r o m a t o r (resolution 0.5 cm -1 at 488 nm) and detected by an RCA C31034 Ga-As photomultiplier tube using photon-counting electronics. The optics, geometry and other characteristics of this apparatus are described elsewhere [11]. Solid samples were held in capillary tubes or contained in cylindrical sample holders (5 mm inner diameter) m o u n t e d coaxial to the laser beam. Solution samples (0.5 ml) were held in the cylindical cuvettes. A scan rate of 100 cm-1/min was em-

137 ployed to record the spectra which were obtained at ambient temperatures. The ultraviolet absorption spectra of protein samples taken before and after prolonged laser beam exposure were identical, indicating that no major structural changes occurred upon irradiation. In recording emission spectra of dilute protein solution (approx. 0.1 mM) it was found that the Raman spectrum of the solvent H20 obscures the SD4-, 7F4 transition while in 2H~O solution the solvent Raman peak interferes with the observation of the SD4 -~ 7Fs emission. By recording spectra separately in both solvents a complete emission profile can be obtained. It should be noted, however, that this interference from the solvent Raman scattering can be circumvented by the e m p l o y m e n t of time-resolved spectroscopic techniques. That is, by using a pulsed laser source along with a variably gated detection apparatus, the instantaneous Raman signal can be temporally displaced from the long-lived signal of interest. Under these conditions, the lower limit of detection of lanthanide ion luminescence is constrained only by the absolute radiant sensitivity of the photomultiplier employed. For the commercial continuous wave laser system employed here for the study of Tb(III) emission spectra, however, we found approx. 0.1 mM to be the useful Tb(III) ion concentration minimum for obtaining-high quality spectra. Materials. The crystalline solid samples were prepared and characterized b y X-ray p o w d e r diffraction as described elsewhere [5]. The chelate complexes and protein solutions are also as previously described [5] (see also figure legends). Results and Discussion SD4 -, 7Fj emission The 488 nm line of the argon ion laser is fortuitously matched to the 7F 6 -* 5D4 absorption of the Tb(III) ion. It is the SD4 state which luminesces, resulting in a series of emission bands corresponding to the SD4 -, 7F6, 7Fs, 7F4, 7F3, 7F:, 7F1, and 7F 0 transitions to the ground 7F-term manifold. The intensities of the various emission bands differ with the SD4-, 7Fs band being the most intense. In most cases n o t all of the bands are detectable. Since we are directly exciting the SD4 level from the 7F 6 ground state, the SD4 -, 7F6 emission is n o t easily observable in our experiments. Fig. 1 shows emission spectra characteristic of two typical crystalline solids (terbium(III) (ethylsulfate)3 • 9H~O and Na3Tb(DPA)3 • 15H20; DPA, dianion of dipicolinic acid), a chelate complex in solution (Tb(III) (DPA), (1 : 3), pH 6.0) and Tb(III) bound to a protein (EF site of parvalbumin). Several features are evident. The positions of the various J -~ 3~ transitions are virtually constant from sample to sample. This is to be expected since the separations between individual J states are dependent on the magnitude of the spin-orbit coupling constant of the central ion which will n o t change significantly with minor environmental changes. The relative intensities of the various J - * J' bands vary from one to another, b u t in qualitatively the same manner from system to system. This intensity variation has been explained theoretically in a semiquantitative manner using the theory developed independently b y J u d d [12] and Ofelt [13] and recently applied to Tb(III) b y several groups [14--16]. In agreement with experiment, the theory

138

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ENERGY (cm -I ) Fig. 1. L u m i n e s c e n c e e m i s s i o n s p e c t r a (5D 4 -~ 7 F j ) t r a n s i t i o n s of solid [ T b ( H 2 0 ) 9 ] ( e t h y l s u l f a t e ) 3 ; solid N a 3 T b ( D P A ) 3 • 1 5 H 2 0 , a s o l u t i o n c o n t a i n i n g [ T b ( D P A ) 3 ] 3-, 1 raM; a n d p a r v a l b u m i n to w h i c h o n e e q u i v a l e n t of T b ( I I I ) h a s b e e n a d d e d (0.1 raM, 0 . 0 5 M p i p e r a z i n e b u f f e r , p H 6.5, 15 5 0 0 - - 1 7 5 0 0 e m -1 in 2 H 2 0 s o l u t i o n , 17 5 0 0 - - 1 9 0 0 0 c m -1 in H 2 0 s o l u t i o n .

predicts the SD4-~ 7Fs transition to be most intense [14]. The transitions to the adjoining 7F6 and 7F4 states are calculated to be next highest in intensity with the remaining transitions predicted to be quite weak. The utility of this theory in its present stage of development in furthering our understanding of the details of the coordination environment of the metal ion is unclear and it will n o t be discussed further here. Of principal interest is the fact that the individual J-~ d bands exhibit considerable fine structure and that there are marked differences in going from system to system. It is this environmentally sensitive fine structure that we wish to exploit as a probe of the immediate coordination environment of the Tb(III} ion. In what follows we will be focussing principally on the most intense SD4-+ 7Fs transition since for experimental reasons, it is likely to be the most useful.

Source o f the fine structure The splitting of the various J -~ J' bands is due to ligand field effects. Ligand field splittings in lanthanide complexes are much smaller than those of their transition metal counterparts, however since the electronic bands are themselves much narrower, these small perturbations are readily observable. Consider the SD4-~ 7Fs transition shown in Fig. 2 for terbium chloride hexahydrate which contains the [Tb(III)Cl~(H20)6] + coordination unit. The acceptor level (TFs) in low-symmetry can be split by the ligand field into as many as 11 levels (2d + 1). At the same time the emitting level (SD4) is comprised of as

139

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F i g . 2. T h e S D 4 -~ 7 F 5 e m i s s i o n b a n d o f T b C I 3 • 6 H 2 0 . T h e s o l i d c u r v e c o r r e s p o n d s t o the s p e c t r u m a t 3 0 0 K w h i l e t h e b a r s i n d i c a t e t h e relative i n t e n s i t i e s o f t h e t r a n s i t i o n s o b s e r v e d at 2 K as a s s i g n e d b y Singh [17].

many as nine levels ( 2 J + 1). Considering the long lifetime of the emitting SD4 level (0.5 ms) at room temperature, there will be thermal equilibrium amongst, and considerable thermal population of, all of the nine c o m p o n e n t levels (kT~ 200 cm -1) which have a total spread of approx. 100 cm -1. Thus, in principle, the SD4 -* 7Fs band can consist of as many as 11 × 9 ~" 99 individual transitions) w i t h o u t any consideration of vibrational levels). Since at high temperatures emissions will occur from the various ligand field-split excited state levels, considerable simplification can be achieved by going to very low temperatures. Singh [17] has recorded the emission spectrum of terbium(III) chloride hexahydrate at 2 K where only the lowest ligand field c o m p o n e n t of the split SD4 state is populated. In this case the SD4 -~ 7Fs transition consists of the expected 11 lines indicated as bars in Fig. 2. Clearly the room temperature spectrum of this substance (Fig. 2) consists of additional lines including peaks at higher energy than the highest energy peak observed at 2 K. This supports

140 the interpretation of the fine structure in these bands as involving ligand field splittings of both ground and excited state levels. Thus, even in cases of relatively high symmetry, a detailed analysis of the observed room temperature fine structure in terms of a ligand field model would be a difficult undertaking. For this reason we will treat the observed fine structure as a spectral signature or fingerprint rather than as data amenable to a detailed theoretical analysis. It is important to realize, however, that the band structure has its origin in ligand field splittings which are dependent on the details of the immediate coordination environment of the metal ion.

Survey of solid state and solution results In order to determine the degree of environmental sensitivity of Tb(III) emissions to change in the metal coordination environment, we have studied a number of structurally well-characterized crystalline solids as well as the aqua ion and several model chelate systems in solution. We have concentrated our attention on the most intense SD4 -* 7F s transition. The results for the solids are shown in Fig. 3 and for the solutions in Fig. 4. Several general features are evident. Although all of the spectra were recorded at ambient temperatures, the crystalline solid samples exhibit better resolution of fine structure features than is true for the solutions. The spectra of the latter, however, are by no means devoid of structure. It is likely that the stereochemical non-rigidity of lanthanide complexes in solution causes some degree of obliteration of fine structure. It is expected that the chelate complexes are fluctional on a time scale short compared to the excited state lifetimes so that their emissions represent a time-averaged sampling of the various fluctional forms present in solution. In the solid state such structural interconversion is absent. From the data at hand few if any generalizations can be made regarding the relationship between the solid-state structure and the spectral characteristics of the SD4 -~ 7F 5 emission band. Panel A of Fig. 3 shows the results for eightcoordinate structures while panel B of the same figure depicts the emissions from nine-coordinate species. While the individual spectra are clearly distinct from one another there are no features which appear to correlate with coordination number. Nor can significant trends be noted with regard to the chemical nature of the coordinating atoms. The spectra set out in Fig. 3 belong to substances which involve the coordination of various numbers of oxygen, nitrogen, and chloride donor atoms, however no evident features correlate with the compositions of the first coordination sphere. Considering the complex origin of the emission bands, it is prudent at present to consider them to be fingerprints characteristic of the various complex and distinct coordination environments. Changes in the coordination environment can be effectively monitored via changes in the emission spectra. For instance, in Fig. 4 the spectra of Tb(III) : EDTA complexes (1 : 1 and 1 : 2) are clearly different indicating a definite interaction with the Tb(III) of the second equivalent of this ligand when it is added to solution. It should be possible to follow the titration of Tb(III) ions in solution with various ligands using this technique. Another application might be for the study of mixed ligand complex formation and for monitoring hydrolytic equilibria via titrations.

141 B

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F i g . 3. T h e 5 D 4 e m i s s i o n b a n d s o f solids o f k n o w n s t r u c t u r e . T h e c o m p o u n d s in t h e l e f t p a n e l (A) are e i g h t - c o o r d i n a t e a b o u t T b ( I I I ) w h i l e t h o s e in t h e r i g h t p a n e l (B) a r e n i n e - c o o r d i n a t e . [ ] , c o o r d i n a t i o n u n i t s : { }, t h e s t o i c h i o m e t r y o f a p o l y m e r i c s t r u c t u r e .

Solid state/solution state comparisons are interesting. For instance, the solidstate emission spectrum of the dipicolinic acid complex Na3Tb(DPA)3 • 15H20 (Fig. 1) is quite similar to a solution spectrum of Tb(III) and DPA present in a 1 : 3 ratio (Fig. 1). This suggests a very similar structure for the complex anion [Tb(DPA)3] 3- in solution and in a crystalline environment. This result is consistent with our finding from lifetime measurements [5] that no water molecules are coordinated to the Tb(III) ion either in solid or solution samples. Furthermore, a nine-coordinate structure very similar to the solid state configuration [18] has been deduced from NMR measurements in solution on other [Ln(DPA)3] 3- complexes [19]. In contrast to the results just discussed for the DPA complexes, the emission spectra of nitrilotriacetate complex are quite different-in the solution and in the solid state (Figs. 3 and 4). These differences, however, are to be expected since in the solid state the Ln(III) ion achieves maximal coordination through bridging o f carboxylate groups to different metal ions and the coordination of only two water molecules [20]. In dilute solution a monomeric, tripod-like

142

coordination of the nitrilotriacetate ligand is to be expected and our luminescence lifetime measurements [5] suggest the coordination of five water molecules in addition to the four-coordinate chelating ligand. The solution and crystalline state coordination environments are very different in this case and this difference is reflected in the striking differences between the respective emission spectra.

Characterization o f metal-binding sites in proteins A major objective of the present paper is to demonstrate the sensitivity of Tb(III) emission structure to differences in metal-coordination sites between different proteins. As an example of the use of Tb(III) emission to distinguish between the metal-binding sites of different proteins we present the emission spectra of Tb(III) bound to the EF site of parvalbumin and to the Ca(II) site 1 of the endoproteinase thermolysin (Fig. 5). From the X-ray crystallographic [22] and sequence [23] results on the native protein, it is known that EF Ca 2÷ is coordinated to the side chain carboxylate groups of Asp-90, Asp-92, Asp94, Glu-101 and to the peptide carboxyl oxygen of Lys-96 as well as to a water molecule. The EF Ca 2÷ is eight-coordinate. It is well established that addition of Tb(III) to thermolysin in the presence of excess Ca(II) (1 mM) results in

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Fig. 4. T h e 5D 4 -* 7F 5 e m i s s i o n b a n d s of T b ( I I I ) s o l u t i o n s . E G T A . [ e t h y l e n e b i s ( o x y e t h y l e n e n i t r i l o ) ] t e t r a a c e t i c acid; N T A , n i t r i l o t r i a c e t a t c . T h e r a t i o s i n d i c a t e d are t h o s e o f l i g a n d to m e t a l . Fig. 5. T h e 5D 4 --~ 7F 5 e m i s s i o n b a n d o f T b ( I I I ) in t h e E F site o f p a r v a l b u m i n (0.1 m M , 0 . 0 5 M piperaz i n e b u f f e r , p H 6.5) a n d c a l c i u m site 1 of t h e r m o l y s i n (0.1 m M , 0 . 0 5 M Tris b u f f e r , p H 7.5).

143 Tb(III) binding to calcium site 1 of the 1,2 double site [8,21]. When Ca(II) is b o u n d in this location in native thermolysin, it is coordinated by side chain carboxylate oxygen atoms of Asp-138, Glu-177, Glu-185 and Glu-190 as well as to a molecule of water [24]. A similar coordination of Tb(III) is to be expected although some rearrangement will occur as the necessary result of the elimination of three carboxylate bridges u p o n the expulsion of the Ca 2÷ at site 2. The emissions of Tb(III) occupying site 1 of thermolysin (Fig. 5) are clearly distinguishable from those arising from this ion b o u n d to the EF site of parvalbumin, demonstrating the sensitivity of this m e t h o d to subtle differences in coordination geometry. The present finding that Tb(III) emissions are responsive to the coordination environment in proteins contrasts with the results of Gafni and Steinberg [9] who found an identical emission spectrum for Tb(III) b o u n d to transferrin and conalbumin. In light of our findings, this would seem to indicate an extraordinary similarity in the metal-binding sites of these otherwise dissimilar proteins. An examination of the circularly polarized luminescence, CPL, of proteinb o u n d Tb(III) would appear to have even greater potential for information content and the detection of subtle structural differences than do the simple emission spectra considered here. Unfortunately judging from the studies reported to date, this expectation has n o t been realized. The CPL studies reported by Gafni and Steinberg [9] and by Brittain et al. [10] reveal that n o t all Tb(III) emission leads to CPL and when it does only t w o different CPL patterns (mirror images of each other) are observed for the SD4 -~ 7Fs transition (the only transition of sufficient intensity to be studied b y this technique). It appears, at the present stage of experimental development, that the use of direct laser excitation of Tb(III) and the examination of the resulting emission under high resolution, is perhaps the most sensitive m e t h o d for detecting changes in the environment of Tb(III) b o u n d to macromolecules. Conclusions

Tb(III) ion luminescent emission (particularly the SD 4 ~ 7F s transition), when examined under high resolution, provides a spectral signature characteristic of the coordination environment of the metal ion. Results on a series of crystalline solids and metal chelates in solution reveal the sensitivity of this technique to environmental changes. The 488 nm line of an argon ion laser provides a convenient excitation source with an intensity sufficient to produce good signal to noise levels on dilute protein solutions (~<0.1 mM). Use of a visible laser eliminates problems arising from protein ultraviolet absorption and photosensitivity. Differences in the coordination environments of Tb(III) b o u n d to the EF site of parvalbumin and to calcium site 1 of thermolysin are clearly revealed in the emission spectra. Acknowledgements This work was supported b y the National Institutes of Health through grant GM23599. We thank Ms. J.L. Morse for supplying the protein samples. We are indebted to Professor W.B. White and Dr. R.P. Burgner of the Materials Research Laboratory, PSU, for enabling us to use the Ramalog instrument.

144

References 1 2 3 4 5 6 7

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Nieboer, E. ( 1 9 7 5 ) Struct. B o n d i n g (Berlin) 22, 1 - - 4 7 R e u b e n , J. ( 1 9 7 5 ) N a t u r w i s s e n s c h a f t e n 62, 1 7 8 - - 1 8 7 Ellis, K.J. ( 1 9 7 7 ) Inorg. Perspect. Biol. Med. 1 , 1 0 1 - - 1 3 5 Switzer, M.E. ( 1 9 7 8 ) Sci. Progr. Oxf. 65, 1 9 - - 3 0 H o r r o c k s , W. deW., Jr. a n d S u d n i c k , D.R. ( 1 9 7 9 ) J. Am. Chem. Soc. 101, 3 3 4 - - 3 4 0 Hor~ocks, W. deW., Jr., S c h m i d t , G.F., S u d n i c k , D.R., Kittrell, C. a n d B e r n h e i m , R.A. ( 1 9 7 7 ) J. Am. Chem. Soc. 99, 2 3 7 8 - - 2 3 8 0 H o r r o c k s , W. deW., Jr., S u d n i c k , D.R., S c h m i d t , G.F. a n d Morse, J.L. ( 1 9 7 8 ) in Rare E a r t h s in M o d e r n Science a n d T e c h n o l o g y ( M c C a r t h y , G.J. a n d R h y m e , J.J., eds.), pp. 1 2 1 - - 1 2 7 , P l e n u m Publishing Co., New Y o r k H o r r o c k s , W. deW., Jr., H o l m q u i s t , B. van Vallee, B.L. ( 1 9 7 5 ) Proc. Natl. A c a d . Sci. U.S. 72, 4 7 6 4 - 4778 Gafni, A. a n d Steinberg, I.Z. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 8 0 0 - - 8 0 3 Brittain, H.G., R i c h a r d s o n , F.S. a n d Martin, R.B. ( 1 9 7 6 ) J. A m . Chem. Soc. 98, 8 2 5 5 - - 8 2 6 0 F r e e m a n , S.K. a n d L a n d o n , D.O. ( 1 9 6 8 ) The Spex S p e a k e r 8, 1--6. (See also: F r e e m a n , S.K, ( 1 9 7 4 ) A p p l i c a t i o n s of Laser R a m a n S p e c t r o s c o p y , p p . 3 8 - - 6 6 , Wiley-Interscience Publications, New Y o r k ) J u d d , B.R. ( 1 9 6 2 ) Phys. Rev. 1 2 7 , 7 2 0 - - 7 6 1 Ofelt, G.S. ( 1 9 6 2 ) J. C h e m . Phys. 3 7 , 5 1 1 - - 5 2 0 Hoshina, T. ( 1 9 6 7 ) J a p . J. Appl. Phys, 6, 1 2 0 3 - - 1 2 1 1 C~Lrnall, W.R., Fields, P.R. a n d R a j n a k , K. ( 1 9 6 8 ) J. Chem. Phys. 49, 4 4 1 2 - - 4 4 2 3 Carnall, W.T., Fields, P.R. a n d R a j n a k , K. ( 1 9 6 8 ) J. Chem. Phys. 49, 4 4 4 7 - - 4 4 4 9 Singh, S. ( 1 9 5 7 ) Ph.D. Thesis, J o h n s H o p k i n s University. (See also: ( 1 9 6 8 ) S p e c t r a a n d E n e r g y Levels o f Rare E a r t h Ions in Crystals (Crosswhite, H.M. a n d Crosswhite, H., eds.), Interscience, New Y o r k ) A l b e r t s s o n , J. ( 1 9 7 2 ) A c t a Chem. Scand. 26, 1 0 2 3 - - 1 0 4 4 D o n a t o , H. a n d Martin, R.B. ( 1 9 7 2 ) J. Am. Chem. Soc. 94, 4 1 2 9 - - 4 1 3 1 Martin, L.L. a n d J a c o b s o n , R . A . ( 1 9 7 2 ) Inorg. Chem. 11, 2 7 8 5 - - 2 7 8 9 Morse, J.L. ( 1 9 7 9 ) M.S. Thesis, The P e n n s y l v a n i a State University Kretsinger, R.H. a n d N o c k o l d s , C.E. ( 1 9 7 3 ) J. Biol. Chem. 2 4 8 , 3 3 1 3 - - 3 3 3 4 Coffee, C.J. a n d B r a d s h a w , R . A . ( 1 9 7 3 ) J. Biol. Chem. 2 4 8 , 3 3 0 5 - - 3 3 1 2 M a t t h e w s , B.W. a n d Weaver, L.H. ( 1 9 7 4 ) B i o c h e m i s t r y 13, 1 7 1 9 - - 1 7 2 5