The Conversion of Fibrinogen to Fibrin. XVI. Electrical Birefringence of Fibrinogen and Activated Fibrinogen' BY IGXACIO TIXOCO, JR.? RECEIVED NOVEMBER 24, 1 R . i l The electrical birefringeiice of fibrinogen and activated fibrinogeti has bceii studied iii the PI-I ratigc from l i to 1 0 i i i a i i r e ; ~ water-glycerol solvent using a single electrical rectangular pulse method developed by Benoit. The hirefriiigencc was tiiiplayed a s a photomultiplier signal on a n oscilloscope screen. From the measured Kerr coefficieiits and from the shapes of the curves for rise and decay of birefringence, dipole moments and rotary diffusioii coefficieiits for the molecules were dctermined. T h e average rotary diffusion coefficient was 36,000 sec.-l, in satisfactory agreement with the results of flow hirefriugence measurements. T h e dipole moment of fibrinogen was calculated by Benoit's theory t o be approximately zero from pH 6 t o 7, t o rise t o a maximum of about 500 D at pH 8.5, then t o decrease again with increasing PH. The Kerr coefficients and therefore t h e dipole moments were sensitive t o small amounts of ions left in the protein solution after dialysis; an increase in electrolyte concentration caused a decrease in the calculated dipole moment. The dipole moment of activated fibrinogen was greater than t h a t of fibrinogen b y an amount which varied with the pH. A maximum increase i n the absolute value of the dipole moment on activation of 110 D was found. This difference, together with the fact that about 10 charges are lost on activation, provides evidence t h a t the activation of fibrinogen occurs a t a site near the center of symmetry of the molecule.
Introduction When thrombin acts on fibrinogen, two peptides are split off,3 with a loss of 10 to 14 negative charges4; since only one collision appears to be involved,j i t may be inferred t h a t the alteration of charge density is confined to a relatively small site on the fibrinogen molecule. The new electrostatic configuration of the activated fibrinogen probably guides the subsequent polymerization, in which the rod-like fibrinogen units are believed6 to undergo lateral dimerization with partial overlapping. Such a pattern can be readily visualized following loss of a group of negative charges either from one end or from the middle of one side of the fibrinogen rod,' but there has been no evidence concerning the actual location of the site of attack by thrombin. It should be possible to draw some conclusions about the latter by determining the change in dipole moment accompanying activation. The classical method of dielectric constant measurements is difiicult to apply for this purpose; although fibrinogen can be dissolved in moderately concentrated urea solution with no added electrolyte, it would not be easy to reduce the conductivity enough to control electrode polarization.s Moreover, recent interpretations of the dielectric increment in protein solution^^^^^ have cast doubt on the usual methods (1) T h i s is P a p e r 33 of a series nn "The F o r m a t i o n of Fibrin a n d t h e Coagulation of Ulood" f r o m t h e lrniversity of Wisconsin, supported in p a r t b y research g r a n t s f r o m t h e Piational I n s t i t u t e s of H e a l t h . Puhlic H e a l t h Service. T h i s w o r k was also supported in p a r i by t h e Otfce of N a v a l Iiesenrch. ITnitFd S t a t e s N a v y . u n d e r C o n t r a c t N i o n r 28509, a n d h y t h r R r w n r c h C o r n m i t t r e o f t h e (;mdtiatr Schnol n f t h r I l n i vernity o f \Viscr,ncin f r o i n f i i n d , s u p p l i e d by t h c \Viicnnsin .\lumni Research I ' r iiiiv,n ~IILI 11 \i'ci11lci, O L ' J L / ~ LiroihhyS (I!J>I).
for calculating dipole inoments. However, electrical birefringence provides an alternative method for measuring dipole moment. From measureineiits of electrical birefringence in response to square pulses, the dipole moment can be determined as well as the rotary diffusion coefficient and additional optical information, according to the theory elaborated by Benoit. l1 Fibrinogen is particularly suited for these rneasurcnients because it is a well characterized, monodisperse protein. Such experiments are described in the present paper; they provide evidence concerning the site of attack by thrombin on fibrinogen, as briefly reported in L: recent note. l 2
Materials The fibriiiogeii w c i q refractionated from Arinour bovine Fraction I, preparation L-210, b y ammonium sulfate, t o give the fraction designated I-L irl Paper X of this series.13 Fibrinogen assays were made h y the nicthod of Morrison." T h e thrombin used was a hovinc preparation (90.51341) containing 32 units/mg., furnished LIS through the kindness of Dr. E. C. Loomis of P a r k , D'tvis and Company. The urea, a Mallinckrodt Analytical Reagent, was used without further purification. Fibrinogen solutions in the urcR-water-glycerol solvents were made by mixitig stock solutions all containing 3 11.1 urea. The concentration of glycerol was determined by a density mcasurement of the aqueous glycerol used in making the stock solution. The comparison of the fibrinogen and activated fibrinogen solutions was made RS follows. A volume of thrombin solution was added t o half of the fibrinogen stock solution, pH about 6.5, and an equ:tl volume of water was added t o the other half. Both solutions were set t o dialyze a t rooin tcinlw-ature ng:tinst 0.1-7 I f sodium chloride. About itti hour after a firm clot had hccn f o r ~ n e d , the ' ~ dialysis h t h 3 .If urea and the dialysis was continuecl f u r a t 3'. The filirin dissolved in the urca t o givc ;t w l u t ion of activated fitit-inogeti, Althougli thew wlutioll\ polymci-izecl t o fortn so!uhle polymers i l l thc PI I r:liigc From X t o 9..i, Kcrr cffrct nic;tsurenlcnti co11kl < t i l l tic made i n this pl1 range because t h e prcsence of g1yc.c.1-01, a clotting retarder," kept any appreciable amounts of polymer from forming. Any polymer with a t least 3 times thc length of fibrinogen would not be detected in the Kerr effect measurements as it would not have time t o rotate perceptibly in the 1 millisecond pulse time used.
ELECTRICAL BIREFRINGENCE OF FIBRINOGRN AND ACTIVAT~TI FIBRINOGRN
July 5 , 1955
Traces of ions left in the solutions after dialysis caused irreproducibility in the Kerr effect measurements. Therefore in one experiment all solvents were ion exchanged using Amberlite IR-120 cation-exchange resin and IRA-400 anionexchange resin (Rohm and Haas). An intrinsic viscosity measurement and fibrinogen assays made a week apart on the same solution indicated t h a t no appreciable denaturation occurred in salt-free 3 M urea.
Method The apparatus is patterned after t h a t of Benoit." A rectangular electrical pulse is applied to the solution illuminated between crossed nicols. The resulting light transmitted by the analyzing nicol is displayed as a photomultiplier signal on an oscilloscope screen. Optical.-The optical bench is a polarimeter on which the position of the analyzer can be read to 0.01 degree. The light source is a 6-8 volt automobile headlight bulb connected to an 8 volt storage battery. The Kerr cell has microscope cover glass windows to avoid depolarization of the light and 18K gold-silver alloy electrodes, 8.73 cm. in length and spaced 0.202 cm. apart. An RCA 1P21 photomultiplier tube detects the light signals. The oscilloscope screen is photographed by a Voigtlander 35 mm. camera with a n f/1.5 lens using Kodak Linagraph Pan film. Electrical.-The pulse generatorIg produces a 0.5 t o 1.5 millisecond rectangular pulse of 25G350 volt amplitude across a 1000 ohm load. The circuit uses a 12AT7 twin triode as a univibrator whose output pulse is applied to the grids of two GYGG tubes. The amplified pulse is connected t o the electrodes in the Kerr cell through a 525 microfarad photoflash capacitor. The rise and decay times of the pulse are each 4 microseconds or less. The output of the photomultiplier cannot be connected directly t o the oscilloscope because a t the desired sensitivity a n excessively long response time is obtained. Therefore the photomultiplier output is connected to the oscilloscope through a 6C4 triode used as a cathode follower. This combination gives a 10% to 90% response time of 25 microseconds with a 1 megohm load resistor across the photomultiplier output. The oscilloscope is a Du Mont 304A Model whose sweep is synchronized with the pulse. A calibrated time base is provided by brightening the oscilloscope trace with an audio oscillator, a Berkshire Labmarker and a n amplifier. Calculations.-Enlarged tracings were made of the photographs of the oscilloscope screen. The Kerr coefficient was calculated from the birefringence curve, using Benoit's" method of comparing light intensities. The electric field strength was determined by comparing the height of the cell voltage pulse with a 100 volt calibrating voltage present on the oscilloscope panel. The rotary diffusion coefficient ( 8 ) was always obtained from a picture showing only the decay of the birefringence, using an expanded sweep. Since the equation of the decay curve is y = e - l 2 O f the slope of a plot of relative height versus time on semi-logarithmic paper gave 8. T o correct the value to that for water a t 20", the viscosity of the solvent was determined with a capillary viscometer, the density having been measured by a Westphal balance. The value of a ,a measure of the orienting torque, was obtained by comparing the experimental curve of the rise of the birefringence with theoretical curves.1' The Kerr coefficient, B = A n / A E 2 , is related to niolecultrr parameters bi- the following equation
c 1' A
n
el P p
k
=
(27rV/15A?z)~g1 - g.j(l'
T = absolute temp. All experiments were made a t room temp., an av. value of 298"A. was used Q = - gi"'/kT = vol. of protein molecule = 38.7 X
for fibrinogenz0 glo - gz0 = a factor involving the principal tliclectric constants of the protein molecule. If the assumption is made t h a t these dielectric consta!its are small compared t o that of the solvent, the factor reduces t o 0.0775~0 ea = dielectric constant of solbent calculated from the table for aqueous glycerol solutions in the ACS hfotiogrdph, "Glycerol."z1 and Wyman's dielcctric incrnnent of 2.69 for urra in water22 I,
Results The Kerr coeficientsZ3 obtained are given in Tables I and 11. The specific conductance of the solutions is also included, as the measured Kerr coefficient depends on the excess electrolyte presTABLE I KERRCOEFFICIENT AND DIPOLEMOMENT OF FIBRINOGEN AS A FUNCTION OF pH Fibrinogen ( 7 g./l.) in 3 M urea in a solvent of 64.0 weight per cent. glycerol in water. Experiment
vol. fraction of protein in s o h . calcd. using the partial specific vol. (0 = 0.71) of fibrinogen = wave length of light. A value of 5,110 A. was taken as the average for the white light used = refractive index of soln. measured with an Abbe refractometer - g.' = an optical factor which will be discussed in a following section = (p/kT)' = dipole moment, assumed parallel to the long axis of the molecule = Boltzmann constant
(18) According t o t h e t h e o r y of Benoit (ref. 11). (19) T h e . pulse generator was desianed a n d t e s t e d b y h f r M hliirray of t h c Ilnivrrsity of &'isconsin P h y s i r s D e p a r t m e n t .
pH
K 11
7.09
K 13
7.63 8.11 8.38 8.60 8.88 9.37" 9.52 8.70 9.13 9.626 6.69 6.73 7,62* 7.95 9.17
K 12
K 11
K 10
x 105 (mhu/cm.) 4.6 5.9 5.7 6.0 6.1 6.2 7.0 7.4 3.6 3.8 5 .1
.. , .
..
B
x
10'
(crn./vult2)
0 . 12 .28 .44 .42 .40 ..12 .31 ',-
. a t >
0.91 .86 .76 0.29 .25 .43
(51 (0) 84 250 230 220 230 140 150 470 450 410 (0)
(0) 2.10
..
.72 .76
390 410
6.18 7 , 00
1.3
0.32
..
. 87
(0) (0)
7.78
0.4 5.7 fj . 8 7.2 7.8
,60
9.50 8.78 9.17 9.63
+ 0)
=
2 1i7
6.70' 7.34'J 7.41 7.48O 7.48"I
, .
.42 ,
5s 451
.:jf;
0.22 .9; .:33 .XI .25 -%
..
290 2::o 331 280 180 (0)
.. 150 120 ..
K9
6.02O , . 0.43 (0) 7 . 50h .. 0.24 81 Cell voltage not measured. Fibrinogen 6.96 g./l., 63.7 weight per cent. glycerol. Fibrinogen 7.1 g./l. 5.0 X M NaCl added. Electrodialyzed fibrinogen. 6.67 X lo-' M NaCl added. 0 Fibrinogen 6 g./l., 64.3 weight per cent. glycerol. * Fibrinogen 6 g./l. (20) S. S h u l m a n , THISJ O U R N A L , 7 6 , 5846 (1953). (21) C . S. M i n e r a n d N. N. D a l t o n , E d i t o r s , ''Glycerol,'' Reinhold Publ. C o r p . , New York, N. Y.,1953. (22) J. W y m a n , Jr., THIS J O U R N A L66, , 4116 (1933). (23) T h e s e coefficients are assumed t o be positive. I n o n e experim e n t a t pH 7.62 i n 2 M u r e a , t h i s a s s u m p t i o n was verified b y use of a q u a i t e r wave p l a t e
TABLE I1 dipole, while for (Y = m only a permanent dipole KERKCOEFPICIESTS ASD DIPOLEMOMENTS OF FIBRINOGEN mechanism is important. IN 2 Af UREAIS TJ'ATER TABLE IV FiSrinogen B X lo' Experiment pH (g./1,) (cm./volt*) (5, ORIENTATIOS MECHANISM OF FIBRINOGEN AS A FUNCTION OF IB 6.77 PH 8.4 0.70 (0) Fibrinogen (7 g.!l.) in 3 hf urea in a solvent of 64.0 weight 8.4 1.04 7.62 210 per cent. glycerol in water. 0.43 7.88 3.26 250 Experiment fiH a /-'(> 0.65 300 8.06 4.06 K 11 6.18 0 , . 49 7.8 8 7 8.18 530 K 13 6.69 0 2.18 7lJO 9.2 4 1 K 12 0.73 !I 1.111 2' i 9,s x ii I; 11 7.00 ti .. K :3 t i 71 13 I lti ( i J) i; 10 7.41 2 1 .3 8 20 12.ti 2.94 421 i I; 12 i 5 0.67 9.00 12.0 2.44 3ii:l K 12 7.88" 5 9. 0 9.85 11 . i 1.41 201) I; 12 7 . %'j >5 1. s l l i 21 11 2 I ,i7 2911 Ii 13 8.11 >5 2.7 1; 11 8.50 1 -3 0.61 eilt. This salt concentration was not calculated K 13 8.70 1 ~2 2.6 because of lack of knowledge about the conductK 13 8.70" 1 3 ,5 ances of the hydrogen and hydroxyl ions and proK 11 8.78 >3 1.2 tein and counter ions in the mixed solvent of glycK 13 9.00" 3 3.6 erol and water. Kerr coefficients in some other K 13 9.13 2- and e2a,w pFI (sec. (sec. -1) pH Experiment Experiment measurements of depolarization of scattered light. If the optical factor is considered independent of 39,400 K 13c 8.70 34,700 K9" 6.02 8.76 38,100 pH in the same solvent, P and therefore p can be K 10 33,600 K 12 7.41 20,500 K 11 8.78 44,800 calculated a t other values of pH. This value of P K gb 7.70 33,600 K 13c 9.00 33,900 calculated from the Kerr coefficient can be divided K 12' 7.89 R 13 9.13 39,200 by Q to give a value of cy to be compared with that 35,800 I( 12 7.95 I( 12" 33,600 K 12' 9.16 35,800 obtained from the shape of the appearance of the 8.11 ET 12 9.17 33,600 birefringence curve. Table IT' shows this com34,200 K 12c 8.28 33,B!lO R1?d 9.52 44,000 parison between a , dependent only on the shape of 8.dcJ Kll I i 1 9 r , d 9,G8 Kill 32,50!l 44,500 the birefririgence curve, and PIQ calculated from K12c Av. 36,000 the height of the curve, the electric field strength, the composition of the solvent, etc. These values Fibrinogen 6 g./l. in 64.8 weight per cent. glycerol (7 = are mostly in reasonable agreement and lend confi0.1152 poise). 5 Fibrinogen 6.95 g./l. in 60.4 weight per cent. glycerol (7 = 0.0893 poise). c Activated fibrinogen. dence to Eenoit's method. Fibrinogen 6.96 g./l. in 63.7 weight per cent. glycerol (7 The calculated dipole moments listed in Table 1 = 0.1138 poise). are shown in Fig. 1. I t is evident that increasing The values of cy calculated from the shape of the salt concentration decreases the apparent dipole appearance of the birefringence curve are given in moment; the highest values are obtained for the Table 1V ; these quantities indicate the mechanism ion-exchanged solutions. Whether this effect is responsible for the orientation of the protein. For due to an actual change in the charge configuration of the protein by ion binding, a phenomenon assocy = 0 the orientation is entirely due to an induced ciated with the ion atmosphere, or an artifact (24) I . T i n o c o . J r . , P h n Thesis, Vniversity of Wisconsin, 1 9 5 4 . caused by electrode polarization cannot he resolved -1,
Q
(25) C
JOI.RYAI ,
S Hocking, Irl Laskowski, J r , and IT. .4.Srliernga, T H I S 1 4 , 77,; ( 1 ( ! 5 ? )
(21;) E 1' C:rsaz;n, private comniunicat ion
July 5, 1955
ELECTRICAL
BIREFRINGENCE OF rIBRINO(;HN AND .qCTIV.I\TED FIBRINOGEN
from these data. Oncley, et a1.,21found that preparations of mercaptalbumin which had been treated with ion-exchange resins gave much higher dielectric increments than had previously been reported, and that addition of various ions reduced the dielectric increment; this would seem t o indicate that the effect is a t least partly due to ion binding. However, the role of the ion atmosphere is still quite uncertain (C. T. O'Konski, personal communication). A recent extension of Benoit's theory28 shows that, if a small component of dipole moment parallel to the short axis exists, the values of p calculated here are slightly smaller than the true components parallel to the long axis. A large component parallel to the short axis would lead to very different values of a and P/Q as compared in Table I V and is therefore unlikely. I t should be pointed out that a t p H values other than the isoelectric p H only effective dipole moAs~ M y ~ e l has s ~ ~shown, ments can be c a l c ~ l a t e d . ~ a symmetrical ion whose center of charge is not a t the center of rotation of the ion will have an apparent dipole moment equal to that calculated if a neutralizing charge were placed a t the center of rotation. The effective dipole moments of fibrinogen are surprisingly small and indicate a very high degree of electrical symmetry. I t is difficult to try to determine what dissociable groups cause the variation in dipole moment with pH. Groups which have a pK in the PH dispersion range and could be responsible are : imidazole of histidine (5.6-7.0), aamino (7.6-8.4), €-amino of lysine (9.4-10.6), and the phenolic hydroxyl of tyrosine (9.8-10.4).31 TABLE V INCREASEOF KERR COEFFICIENT A N D DIPOLEMOMENT OF FIBRINOGEN ON ACTIVATION Fibrinogen (7 g./l.) in 3 X urea in a solvent of 64.0 weight per cent. glycerol in water. Experiment
B
PH
x
104 (cm./volt*)
lPal 1P"I
K 12 6.73 0.24 K 12-A .27 6.66 7.50" K 9 .24 7.50" K 9-A .29 K 12 7.62' .43 K 12-A 7 .65' .44 K 12 7.95 .72 K 12-A 1.03 7.89 K 13 8.70 0.91 K 13-A 1.16 8.70 K 13 9.13 0.86 K 13-A 1.18 9.00 9.17 K 12 0.76 K 12-A 9.16 .87 K 13 9 . 62b .76 K 13-A 9 . 68b .82 Fibrinogen 6 g./l. Fibrinogen 6.95 per cent. glycerol.
-
(D)
lPB1
+
IPLI
(D)
( 0)
(0)
80
240
4
485
110
890
81
1020
100
1000
40
860
20
840
g./l., 63.6 weight
( 2 7 ) H . Neurath and K.Bailey, Editors, "The Proteins," Academic Press, Inc., New York, N. Y . , 1953, p. 708. (28) I . Tinoco, Jr., THISJOURNAL, in press. (29) E. J. Cohn and J. T . Edsall, "Proteins, Amino Acids, and Peptides," Reinhold Publ. Corp., New York, N. Y., 1043, p. 552. ( 3 0 ) K. J . Mysels, J . Chem. P h y s . , 21, 201 (1953). ( 3 1 ) Neurath and Bailey, ref. 27, p. 477.
I
I
3479
I
I
I
I
7
8
9
PH. Fig. 1.-Dipolc nioincnt of fibrinogen plotted against pH: circles left black, experiment K 13; right black, K 12; top black, K 11; open, K 14.
The change of dipole moment on activation is shown in Table V. Both solutions in each experiment were prepared in exactly the same way, but in the absence of buffer the p H values still differed slightly in some instances; A indicates the activated fibrinogen solution. The change in dipole moment, parallel to the long axis of the molecule, on activation may be written as Ps
- Pu
= xei x , i
where ~i = electronic charge (4.8 X 10-lo e.s.u.), which can be positive or negative; xi = distance from center of of molecule to the location of a charge. The sum is taken over all charges lost or gained on activation. On activation by thrombin, fibrinogen loses two peptides3: peptide A contains 12 dicarboxylic acid groups of which 4 are in the amide form, 1 lysine and 2 arginine; peptide B contains 10 dicarboxylic acid groups of which 3 are in the amide form, 1 lysine, 2 arginine and 1 tyrosine.* Moreover, 1 aamino group is lost and 3 0-amino groups are gained.33 The net change in charge, Az, is there. fore about ten34in the $H range from 7 to 10, but will depend slightly on p H because of the a-amino groups. If it is assumed5 that the alteration of charge all takes place in a relatively small area on the fibrinogen molecule a t a distance d from the center of symmetry, this distance can be calculated (32) The center of symmetry is chosen as the origin so that the neutralizing charges which must be added at this pointm need not be explicitly considered. (33) L. Lorand and W. R. Middlebrook, Scirncc, 118, 595 (1953). (34) Mihalyi' has calculated a net change in charge of 14 from elec trophoresis measurements. The siight discrepancy in these values does not affect the conclusions drawn.
using the equation d =
!wal
* €As
IPUI
Both signs must be included as only the absolute magnitude of the dipole moment can be measured. Since the maximum change in dipole moment observed was eitherol10 or 102G D ,the value of d is found to be < 2.5 A. or 25 A. The result thus indicates t h a t activation takes place near the center of the fibrinogen molecule. A potential source of doubt regarding the interpretation of the low Ap is the possibility that the charge pattern may readjust, through proton migration, to minimize Ap regardless of the site of loss of the negative charges.Id However, it seems unlikely t h a t the result would be SO closely similar a t pH 7, where the migrating protons will be eychanging on histidine residue, and a t pFI 9 10 where they will be exchanging on tyrosiiie and lysine residues. The two peptides have nearly equal charges; therefore it could also be possible that tlic peptitles (?i) C 1 anlurd p r i v a t e
i~,iliiltiintcatiirri
Thermodynamic Properties of the Ammonium Ion BY AUBREYP. ALTSIIULLER RECEIVED FEBRUARY19, 1955
Structural and spectroscopic data 011 the ammonium ion recently have been available which make possible the statistical thermodynamic calculation of the thermodynamic functions C:, (11"- 1 l ~ ) . ' T , - ( F o - H,O)!T and Sofor XHHq+(g). The S-H internuclear distance in NH4+ has been obt:iined from both neutron diffractioiil-3 and nuclear niagnetic resonance m e a s ~ r e n i e n t s . ~ JThe most accurate determination of th,e N-.H distance5 gives a value of 1.0:E rt 0.00.5 A . Thus the moment of inertia, 1,has the value (4.75 f 0.051 X g. cni.2. The vibrational freqiiencies of the spherical top NH4+(T,] symnietry is assunied for h"~+ in the gaseous state) have been found to be ~ ~ ( = 1 )3041 c111.-~; v , ( 2 ) = 1682 c171.-1; v&) = 33090 cn1.-' and yq(.?) = 1403 C I I I . - - ~ iron1 infr:iretf nieasurenients on films of KH4Cl.6 The values of the therinodynamic functions ct, (11"- Ht):T,- (Fo- 11;) and S" for NH4+ (g) from 200 to 1000°K. as calculated by the rigid rotator-harmonic oscillator approximation are listed in Table I for the itleal gas state a t one atmosphere pressure. ( 1 ) G . H. (:oldiicltiiiid ancl D. C;. ITiirit, Phyr.. Re;'.,8 3 , 88 ( l ( L - J ) : 8 6 , 747 ( 1 9 5 1 ) . ( 2 1 H. A . 1,evy a n d S . W. Prtrrsoti ihitf , 86, 7tX (19.-t?). ('3) H. A . I.evy a n d S. LV, Prtrr>
