J. Mol. Biol. (1976) 102, 143-156

A Neutron Small-angle Scattering Study of Bovine Fibrinogen G. MARC~IJERIE

Laboratoire

d’HematoEogie, DRF, Centre #Etudes Nucldaires B.P. 85, 38041 Grenoble, France AND

I~nAitut fiir

H.B.

de Grenoble

STCHRMANN

Physikalische Chemie der Universitiit 6500 Ma&z. Geprnn>q

&!a.inz

(Received 10 February 1975, and in revised form 10 November 1975) Neutron small-angle scat,tering of fibriIlogen in \.arious H20/“H,0 mixtures is strictly proportional to the square of tile contrast at a resolution of less than 15 A. Therefore, the exnt~ scattering densit,y with respect to the solvent is positive everywhere. The maximum scattering density is only a quartcir of that encountered with globular proteins. It is not so low that fibrinogen could be regarded as a statistical coil. The shape is resolved in tcsrlrw of multipoles. d stepwisc increase in the resolution is achieved by taking into account an increasing number of multipoles; on addition of a dipolar term to the spherical average a cup-shaped model results. A still better description is obtained if tile quadrupolar term is derived from t,he scattering curve. The superposition of the first three multipoles leads to a banana-shaped fibrinogen model. This result, from the analysis of the sc:attf:ring pattern in term< of mult,ipoles, is cornpined \vith other physicochemical a rid hioc:hr~nAcal properties of fibrinogen.

1. Introduction Fibrinogen is a plasma protein which is of special interest because it polymerizes after its activation by the enzyme thrombin. It is a dimer which consists of two halves containing three chains, Au, BP and y, held together by disulfide bridges (for a general review see Blomback, 1967; Murano, 1974). Biological and chemical studies have suggested t’he existence of three basic structural zones in this protein. Cnder prolonged plasmic degradation, for imtance, three resistant fragments are released, namely two D and one E. On the other hand, several disulfide bridge-containing fragments (DSK) have been identified after cyanogen bromide treatment, mainly HolDSK and NDSK or HilDSK, which have chemical, immunological and sequence relationships with the fragments D and E (Blomback & Blomback, 1972; Marder et al., 1972). The exact localization of these basic structural and functional fragments has not yet been established. This is partly due to the fact that the shape of the fibrinogen molecule still remains uncertain in spite of the great, amount of work which has been done on this protein (for a general review see Doolit.tle, 1973).

144

G. MARUUEHI

I.: :\SL)

H.

H. STC’HHhlAXE

Since native fibrinogen has not yet, been crystallized, conformational information has mostly been provided by electron microscopy and physicochemical studies on dilut,e solutions. Depending on. the method of investigatIion, different models have been proposed leading to considerable controversy. Suggested shapes include an elongated unhydrated prolate ellipsoid with a length of 690 A (Shulman, 1953 ; Kay & Cuddigan, 1967), a nodular rod-like particle of 450 A length (Hall & Slayter 1959; Bachmann eb al.. 1975) and finally a highly hydrated sphere of 240 A diameter (Koppel. 1966). Therefore, attempts to reconcile t#he results from electron microscopy and from physic0 chemical investigat,ions were more or less unsuccessful and gave no definite molecular shape for fibrinogen. A recent’ tentative proposal was made by Bachmann et al. (1975) and by Ledrrer & Hammel (1975). According to these authors a probable model is a highly hydraeed cylinder of 450 A length and of 90 A diamet#er. This interesting conception of the fibrinogen molecule will be discussed later. Light scat’tering and small-angle X-ray scattering are known to be powerful methods for investigating the shape of proteins in solution. But the results obtained by both methods wit’h fibrinogen solutions were subject, to ambiguous interpretation (Lederer, 1972; Marguerie. 1972). This paper presents a neutron scatt,ering investigation of fibrinogen solut’ions, where special care has been taken to avoid aggregation and enzymatic degradat’ion. Several reasons favour t#he use of neutrons for scattering experiments. At low momentum transfer (K = (&/h)sin 0, 20 = scattering angle? X = wavelength) this technique is easier than X-ray small-angle scattering because the background scattering can be kept relatively low. Furthermore the energy of the incident neutrons is comparable to t,hat, of the t’hermal motIions and is t’herefore most probably harmless to sensitive biological structures. Finally, contrast variation is most, easily achieved in neutron scattering by using H,0/2Hz0 mixtures as the solvent,s. This experimental procedure allows the separation of the scatt,ering function of the shape from that of the internal structure (Stuhrmann, 1970a,b; lbel & Stuhrmann, 1975).

2. Materials and Methods Purified

fibrinogen

( > 98yo clot,table

protein) was obtained from bovine plasma by in sodium citrate ether precipitation (Keckwick et al., 1955). The plasma was collected solution (loo/e) containing 0.025 M-E-amino caproic acid and iniprol, 5 U/ml (purchased from laboratoire CHOAY, France), in order to prevent proteolytic degradation (Bang et al., 1971). The samples were exhaustively dialyzed at 4°C against 6.3 M-KC& and clarified by using the rotor number SW25-1) in a 30 min ultracentrifugation (25,000 revs/mm, Spinco model L preparative ultracentrifuge. The protein concentration was determined coefficient EfA,% by measuring the absorbance at 280 nm, using a specific extinction of 1.59 (Bion et al., 1971). Monodispersity of the solutions was assumed on the basis of light-scattering and viscosity measurements (Marguerie & Suscillon, 1973). Fibrinogen was dialyzed against 12 different H,O/sHsO mixtures (0.3 M-KCl) at room temperature. Above 60% 2H,0 an increase of the turbidity of the solutions was observed indicating slight aggregation due probably t,o a decrease of the fibrinogen solubility. corrected before any scattering measurements This unexplained problem was, however, were made, by increasing the salt concentration from 0.3 ~-Kc1 to 0.5 M-KCl. A series of solutions with decreasing protein concentration C from 20 to 1 mg/ml was prepared in order to extrapolate small-angle scattering to infinite dilution. The neutron-scattering experiments were performed at the high flux reactor of the Institute Max Von Laue-Paul Langevin at Grenoble. The small-angle scattering device

FIBRINOGEN

MOLE(‘UL.41~

I&i

SHAPE

NlLS (I) 1 I) whicll u-o used in o11r experiments is described by Schmatz et al. (1974). Bs, at a givvu tl&octor position, only a part of the scattering curve can be measured. different distances betweort tho sample and the 2-dimensional multidector wert’ chosen (Fig. 1). ‘1‘11~mc*nsuring time was about 10 min for each curve. ‘l’l~ data w.‘ro scaled by using t II

A neutron small-angle scattering study of bovine fibrinogen.

J. Mol. Biol. (1976) 102, 143-156 A Neutron Small-angle Scattering Study of Bovine Fibrinogen G. MARC~IJERIE Laboratoire d’HematoEogie, DRF, Centre...
963KB Sizes 0 Downloads 0 Views