Molecular properties of lamprey fibrinogen.

Application of conventional methods not previously used for preparing lamprey fibrinogen has yielded preparations of very high purity and stability. These preparations made possible a detailed evaluation of molecular weights in normal and chaotropic buffers. The native molecule repeatedly gave a molecular weight of 352,000 to 358,000 via sedimentation equilibrium in citrate buffer; a molecular weight of 354,500 was obtained in guanidine buffer. An anomalous increase of the value of the measured apparent partial specific volume was observed in guanidine buffer. Molecular weight data of the reduced and alkylated subunits of lamprey fibrinogen, obtained by four different methods, have led us to assign molecular weights of 110,000 for (A)alpha, 72,000 for (B) beta, and 50,000 for gamma. Based upon these molecular weights obtained for the subunits, as well as that of the native fibrinogen molecule, the subunit composition can best be fitted to the formulation [(A)n alpha, (B) beta 2, gamma 2] rather than the conventional[(A) alpha 2, (B) beta 2, gamma 2] which would yield a molecular weight of 464,000. Analysis of a stabilized clot induced by Ca2+ showed only gamma dimers; alpha subunit polymerization was undetectable. Cross-linking of lamprey fibrin in the presence of dansylcadaverine and Ca2+ results in fluorescent labeling of the gamma chains and to a lesser extent the gamma dimer. Differing from other reported vertebrate cross-linking systems, the lamprey fibrin alpha subunit appears essentially unreactive in both polymer formation and dansylcadaverine incorporation. These distinct molecular properties may be reasonably attributed to the existence of the single (A)n alpha subunit in the molecular structure of the molecule.

Application of conventional methods not previously used for preparing lamprey fibrinogen has yielded preparations of very high purity and stability. These preparations made possible a detailed evaluation of molecular weights in normal and chaotropic buffers. The native molecule repeatedly gave a molecular weight of 352,000 t o 358,000 via sedimentation equilibrium in citrate buffer; a molecular weight of 354,500 was obtained in guanidine buffer. An anomalous increase of the value of the measured apparent partial specific volume was observed in guanidine buffer. Molecular weight data of the reduced and alkylated subunits of lamprey fibrinogen, obtained by four different methods, have led us to assign molecular weights of 110,000 for (A)a, 72,000 for (B)p, and 50,000 for y. Based upon these molecular weights obtained for the subunits, as well as that of the native fibrinogen molecule, the subunit composition can best be fitted to the formulation [(A),a, (B)& yz] rather than the conventional [(A)aZ, (B)& yz] which would yield a molecular weight of 464,000. Analysis of a stabilized clot induced by Ca2+ showed only y dimers; a subunit polymerization was undetectable. Cross-linking of lamprey fibrin in the presence of dansylcadaverine and Ca" results in fluorescent labeling of the y chains and t o a lesser extent the y dimer. Differing from other reported vertebrate cross-linking systems, the lamprey fibrin a subunit appears essentially unreactive in both polymer formation and dansylcadaverine incorporation. These distinct molecular properties may be reasonably attributed to the existence of the single (A),a subunit in the molecular structure of the molecule.
Lamprey (Petromyron marinus), one of the most primative vertebrate species, has a fibrinogen containing three distinct subunits, ( A ) a , (B)@, and y, (1,2), similar to that found in higher ordered animals in the phylogenetic evolutionary scale (3). This contrasts sharply with coagulating proteins found in nonvertebrates such as "lobster fibrinogen" (4) and the coagulinogen of the horseshoe crab, limulus (5). Certain major lamprey proteins which have been characterized also exhibit * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. distinct differences from those of other vertebrate species, i.e. the subunit composition of the hemoglobin (6) and the immunoglobulins (7). Different molecular weights have been reported for lamprey fibrinogen and for its constituent subunits. A molecular weight of 440,000 was obtained postulating the conventional six-chain structure (8). The molecular weight of the a chains was reported as approximately 105,000 by gel electrophoresis. Lamprey @ chains were reported to be about the same size as human a chains and y chains about the same size as mammalian y chains. Direct measurements on unreduced lamprey fibrinogen gave a molecular weight of about 400,000 (8). In a later publication, the molecular weights of the individual chains as determined by gel electrophoresis were reported as 100,000 for the a chain, 55,000 for the p chain, and 47,000 for the y chain (9). In this same publication, amino acid composition studies on a, @, and y chains from lamprey fibrin lead to a calculated molecular weight of 70,000 for the a chains and a calculated molecular weight of 360,000 for intact lamprey fibrinogen.
The values of the molecular weights of the individual chains as obtained by gel electrophoresis are consistent with other reported values. Molecular weights of 50,000 for y chains, 55,000 for @ chains, 70,000 for (B)@ chains, 110,000 for ( A ) a chains, and 210,000 for a dimer have been reported (10). We have reported molecular weights obtained by gel electrophoresis of 112,000, 68,000, and 50,000 for the three subunits and a molecular weight of 326,000 for the intact molecule by sedimentation equilibrium on a sample purified by alcohol precipitation (2). The possibility of a single A(a) subunit as a means of reconciling the molecular weights of the subunits and the molecular weight of the intact molecule was suggested here. The present study utilizes lamprey fibrinogen of very high purity and stability and addresses itself to a detailed investigation of the properties of this molecule.
In order to perform physicochemical studies in aqueous solutions, we have developed modified methods for the isolation of lamprey fibrinogen in greater yields, high purity, and stability. Extensive molecular weight determinations of the isolated subunits (chains) were made by sedimentation equilibrium in 6 M guanidine buffers, gel fitration in 6 M guanidine buffers on Sepharose, and polyacrylamide gel electrophoresis to resolve these discrepancies. Concurrently, molecular weight determinations of the native and denatured lamprey fibrinogen were also performed by sedimentation equilibrium in citrate-NaC1 and 6 M guanidine hydrochloride buffers. Based upon these studies, we have re-evaluated the molecular weight and subunit constitution of the lamprey fibrinogen.
Concomitant with these investigations, the mechanism of lamprey fibrin cross-linking was re-examined for possible reflection of a subunit influence. The negligible extent of a polymer formation observed in the stabilized plasma clot as well as the in vitro fibrin clot was reconfiied (2, 8). The reactivity of lamprey fibrin a and y subunits to intrinsic crosslinking enzyme (Factor XIII) was studied kinetically by incorporation of dansylcadaverine during fibrin formation.
Our thorough investigation of the molecular properties has brought forth some pertinent observations that lamprey fibrinogen is different from fibrinogens of other vertebrates.

MATERIALS AND METHODS'
Materials -Acrylamide, N,N'-merhylene-bis-aCrylamlde, N . N . N ' . , , -t e t r~e t h y le t h y l e n e d r a m l n e , monxum p e r~u l f a t e , C o c m a s~l e Blue were electrophoresis purlty reagent grade from Blo-Rad.
lVMiOnlum S u l f a t e ( s p e c i a l enzyme g r a d e l , q u a n l d l n e XC1 ( u l t r a p u r e ] , urea ( u l t r a p u r e 1 a n d   'e;..

( 2 )
he measured a~oarent oarrial soeclfic volume as defined above is the proper welrhfs are t o be obrarned 1 2 4 ) .
rhermodynamlc parameter which must be used if correct anhydrous molecular

RESULTS
Yield a n d Purity-Using 400 ml of starting material of either of the designated plasmas, the yield of lamprey fibrinogen, 95% + clottable, was usually in the order of 600 mg. The reduced preparations when examined on 5% or 7.5% SDS'polyacrylamide gels show three distinct bands (Fig. 1, zero time). No (A)a doublet is discernible. Heat denaturation, as evidenced by precipitation, was notable a t 31°C; the entire contents could be precipitated by heating for 30 min at 37°C. No differences were evident when this denatured material was reduced and examined on SDS-polyacrylamide gels. The results were identical with Fig. 1, zero time. Addition of either human thrombin (plasmin free) or lamprey thrombin to solutions of the highly purified lamprey fibrinogen produced a marked change in the mobility of the , L? subunit compared to the (B)P of the fibrinogen, Fig. 1, time 1 min. This has been ascribed previously to the removal of the large carbohydrate containing B-peptide (25). Examination of the fibrin resulting from the action of plasmin-free human thrombin clearly shows the appearance of an a subunit doublet as well as low molecular weight material (See Fig. 1). No mobility differences could be discerned in the y subunits in the native fibrinogen or resulting fibrin formed via human thrombin. It would appear that the fibrin a subunits as well as the ,L? subunits are susceptible to further degradation by the action of human thrombin (Fig. 1). Lamprey thrombin removes a 6-residue A-peptide along with the 36-residue carbohydrate containing B-peptide (26). Our purest lamprey thrombin preparations rapidly clotted lamprey fibrinogen, but even in the presence of Trasylol, the fibrin clots lysed within short periods of time. These lysed preparations, when reduced and examined on SDS-polyacrylamide gels, showed major degradation of the a subunits as well as degradation of , L? subunits. These studies will be reported in more detail in a subsequent publication.
Constituent Subunits- Fig. 2 illustrates the chromatography of the reduced and alkylated subunits on a column of CM-52 in 8 M urea and 0.1 M Tris-acetate buffer with a linear buffer gradient from pH 4.75 to pH 5.80. Identity of the subunit constituents was shown by running aliquots from the various numbered tubes on SDS-polyacrylamide gels. It is notable that the fmt peaks (Tubes 33 and 36) showed the same mobilities as the y-subunits in the controls. In all, eight tubes, as numbered in Fig. 2, were assayed electrophoretically, showing a clear separation of the y, (B)P, and (A)a subunits, emerging in that order. Upon dialysis against distilled water, the y and (B)P subunits rapidly precipitated; the (A)a subunit precipitated more slowly. The horizontal bars in Fig. 2 indicate the combined fractions which were dialyzed and lyophilized. All of the above preparations were completely soluble in 6 M guanidine-Tris buffers.
Amino Acid Compositions-The amino acid composition of our purest preparation is given in Table I  that the molecule has an extremely high glycine content. Performic acid oxidation by the method of Hirs (16) followed by chromatographic analysis gave 49 residues of cysteic acid, indicative of 24 to 25 disulfide linkages. The molecular weight, as determined. by amino acid analysis, is in the neighborhood of 360,000, a figure in close agreement with physical data.
Amino acid analyses of the isolated reduced and alkylated chains are also shown in Table I. These analyses were utilized for the calculation of the partial specific volumes of the native molecule and of the subunit chains according to the method of Lee and Timasheff (22).
Molecular Weight Determinations- Table I1 gives a com-parison of the subunit molecular weights obtained by two methods, SDS-polyacrylamide gel electrophoresis and gel fdtration chromatography on Sepharose CL-4B in 6 M guanidine HC1-Tris buffer (0.05 M ) , pH 7.5. Sedimentation equilibrium of the native molecule and subunit chains in aqueous and guanidine buffers will be presented in detail in a separate section.
Employing SDS-polyacrylamide gels of 5% and 7.5%, we consistently obtained molecular weights of 110,000, 78,000, and 50,000 for the (A)a, (B)P, and y subunits, respectively.
The a subunits of lamprey fibrin resulting from the actioil of lamprey or (plasmin-free) human thrombin, even for relatively short reaction times, resulted in the appearance of a doublet of this subunit (see Fig. 1) with molecular weight of the order of 110,000 and 100,000. In addition, the molecular weight obtained for the / 3 subunit of lamprey fibrin (following Bpeptide removal either by lamprey or human thrombin) was in the order of 66,000 to 68,000, lower than expected for the removal of the 36-residue glycopeptide (21). Quantitation of the gels, assuming that all of the chains bind dye equally, gave the mass ratio of the As shown in Fig. 3, the logistic distribution curve, given by the equation where b and c are fitting parameters, was fitted to the KD and molecular weight values of the standards, and the molecular weights of the lamprey subunits were calculated from the inverse equation   Fitting gel filtration data requires making some assumptions regarding the distribution of pore sizes in the gel. The problems inherent in this have been very well discussed by Rodbard (15). While the most common procedure is to use a linear relationship between KD and log (Mr), it has been shown that this is significantly inferior to using either the log normal or logistic distribution functions. While the log normal and logistic distributions are essentially equivalent, the logistic function is much easier to use computationally since the log normal distribution requires the use of either inverse error functions or integrals which cannot be evaluated analytically.
Sedimentation Analysis: Sedimentation Coefficient-The sedimentation coefficient, s, is defined by the equation where w is the angular velocity of the rotor (radians/s) and r is the radial position of the maximum ordinate. This equation may be integrated to give r = rmexp(w2st) (6) where r, is the radial position of the meniscus. This form has the advantage that s can now be a fitting parameter for the nonlinear least squares fitting of this equation Partial Specific Volume-It is necessary to know the partial specific volume of the protein in order to make the corrections for solvent density in sedimentation velocity studies and to calculate the molecular weight in sedimentation equilibrium studies. We have calculated an apparent compositional partial specific volume based upon the amino acid composition and upon the assumption of 3% carbohydrate by weight with the same partial specific volume as that of glucose. A compositional partial specific volume of 0.703 cm3/g was calculated for the native lamprey fibrinogen where the compositional partial specific volume from the amino acids was 0.706 cm3/g and the carbohydrate partial specific volume was 0.623 cm3/g. This latter value has been calculated from density data in the "Handbook of Chemistry and Physics." A value of 0.708 f 0.004 cm3/g was obtained experimentally for the apparent partial specific volume of lamprey fibrinogen which had been dialyzed to equilibrium against citrate buffer. The concentrations were determined spectrophotometrically using a value of E&5 = 12.3 for the extinction coefficient which was determined from dry weight measurements. The close agreement of this value with that of the calculated compositional partial specific volume suggests minimal preferential interaction with solvent components.

(8)
where cr is the concentration at radial position r, cb is the concentration at the cell bottom, M is the molecular weight, and A is given by where U is the partial specific volume, p is the solution density, R is the gas constant, and T is the absolute temperature. If more than one macromolecular component is present, then In either case, the values of M and cb or the values of M, and Cb,, are fitting parameters which are adjusted by the nonlinear least squares curve-fitting program in MLAB. Our experience with several samples of highly purified lamprey fibrinogen has been that these have been over 90% monomer, and that the highest molecular weight material present has, within the limits of precision of our measurements, always had a molecular weight which was an integral multiple of that of the fibrinogen monomer. Thus, Equation 10 could be written as

The relative quantity of each component is readily obtained by means of equations involving conservation of mass. Thus, c,,(rz -rm2) = lm ct.,,exp[AM(r2 -rb2)]dr2 (12)
Where co,; is the concentration of the ith component before distribution in the centrifugal field. Equation 12 then gives The relative amount of each macromolecular component is then given by The possibility of thermodynamic nonideality has also been considered. The apparent molecular weight is given by the equation ( 16) where the subscripts indicate that the apparent molecular weight is a function of concentration and where B is a virial coeffkient. Equation 11 then becomes  -rb2)/(1 + nBMc,)] (17) and B becomes an additional fitting parameter. Because cr appears on both sides of Equation 17, the root finder in MLAB must be used in order to fit the data when this equation is used.
Equations 8, 11, and 17 may be described as mathematical models for fitting the data from the ultracentrifuge. Three criteria were used in evaluating the fit of a given model to the data. First, the parameters obtained in fitting had to be physically meaningful, i.e. having nonnegative values. Second, a minimum value of the root mean square error denoted a superior fit. Since the root mean square error obtained using MLAB takes the number of degrees of freedom of a model into account, the comparison of root mean square values for models having differing numbers of parameters is valid. Third, the deviation of experimental data points from the fitting line should not exhibit systematic error. If two models satisfy the f i t criterion and have essentially equal root mean square errors, a more random pattern of deviation can become an important selection criterion. Figs. 4 and 5 show examples of the fitting procedure. Fig. 6 illustrates a good example of the deviations from the fitting line. Fig. 7 shows how this can be used to discriminate between two models that have similar root mean square errors.
Six analyses of three preparations of highly purified native lamprey fibrinogen indicated that the solutions did not exhibit thermodynamic nonideality and that they had 3 to 6% tetramer present. The mathematical model we fit had molecular species from monomer through hexamer, subject only to the constraint that all fitting parameters had to be positive or zero. The concentration parameters of all species but monomer and tetramer were either zero or vanishingly small for all cases. Thus, the monomer-tetramer model satisfies the best fit criteria, but does not eliminate the small finite probability of the presence of dimer, trimer, etc. The monomer molecular weight was 352,000 & 12,000 using the calculated compositional partial specific volume of 0.703 cm3/g and 358,000 & 12,000 using the experimentally measured partial specific volume of 0.708 cm3/g.

The molecular weights of the (A)a, (B)P, and y subunits of
lamprey fibrinogen were also measured by equilibrium ultracentrifugation in 6 M guanidine buffer since they were not soluble in the citrate buffer. The apparent partial specific volumes of the subunits in this buffer were calculated from the amino acid composition by the method of Lee and Timasheff (22), assuming an equal weight per cent of carbohydrate of the (B)P and y subunits. This gave calculated apparent partial specific volumes of 0.687, 0.700, and 0.722 cm3/g and resultant molecular weights of 78,000, 57,700, and 50,100 for The considerable disparity between these values and the molecular weights obtained by gel filtration and by gel electrophoresis led us to suspect the validity of the values of these calculated apparent partial specific volumes. We measured the molecular weight of intact lamprey fibrinogen in the 6 M guanidine buffer in order to determine whether this disparity was due to the use of incorrect values for the apparent partial specific volumes or was due to false values obtained with the gel methods. Calculation of the apparent partial specific volume of the lamprey fibrinogen in 6 M guanidine buffer gave a value of 0.693 cm3/g. A molecular weight of 273,000 was obtained using this value for the apparent partial specific volume. Since there was no evidence for degradation of the lamprey fibrinogen in the 6 M guanidine buffer, this strongly

FIG. 7.
Difference plots for comparing the quality of the tits for two different models. Plot 1 shows the distribution for the data points about the fitting line for the equilibrium concentration distribution of lamprey fibrinogen in 6 M guanidine buffer after 7 days at 7200 rpm and 20°C. The best fit requires a thermodynamic nonideality term. This should be contrasted with plot 2 which shows the distribution obtained after another 7 days of centrifugation. No nonideality term was required for the best fit. Although the root mean square errors are comparable for the two fits, the more random nature of the distribution in plot 2 is readily apparent.
suggests that the calculated apparent partial specific volume is incorrect. Calculation of the apparent partial specific volume necessary to give a molecular weight of 352,000, as obtained with the calculated compositional partial specific volume, gave a value of 0.734 cm:'/g. A value of 0.735 k 0.003 cm"/g was obtained experimentally from densimetric measurements on lamprey fibrinogen dialyzed against the 6 M guanidine hydrochloride buffer. An experimentally determined value for the extinction coefficient of EL& = 13.2 was used to determine the protein concentrations. A value of 354,500 f 7,500 was obtained for the molecular weight using this apparent partial specific volume. Application of this apparent partial specific volume to the individual subunits gives molecular weights of 104,700, 71,600, and 54,300 for the (A)a, (B)& and y subunits, respectively.
That this anomalous behavior is not unique to lamprey fibrinogen was then demonstrated when a molecular weight of 282,000 was obtained for bovine fibrinogen in 6 M guanidine buffer using the literature value of 0.718 cm"/g for the partial specific volume (27). In order to obtain the cited value of 330,000 for the molecular weight, the partial specific volume had to be increased to 0.741 cm:'/g, an increase of 0.023 cm3/ g as compared to the increase of 0.031 cm"/g for lamprey fibrinogen. It is of interest to note that solutions of fibrinogen and the constituent chains appeared to exhibit thermodynamic nonideality when insufficient time was allowed for equilibrium. The extent of the nonideality diminished with time and eventually disappeared. Equilibrium was assumed to be obtained when no nonideality could be demonstrated in two successive measurements taken 3 days apart. Times to equilibrium were approximately 2 weeks for the individual chains and 3 weeks for the intact molecule with column lengths of 6 mm. It is also of interest to note that the molecular weights obtained after correcting for nonideality did not differ very significantly from the final values obtained. Cross-linking- Fig. 8 illustrates the inhibition of cross-linking of lamprey fibrin when dansylcadaverine is added to a mixture of lamprey fibrinogen, Cap+, and either lamprey or plasmin-free human thrombin. Only the y chains incorporate the fluorescent amine. It is also possible to discern minute formation of fluorescent y dimer probably caused by isopeptide bond formation prior to complete saturation of the acceptor sites by dansylcadaverine. It should be stressed that no extrinsic Factor XI11 has been added. The a subunit showed no incorporation of fluorescence even after an 8-h incubation of the reaction mixture. These results are in contrast to similar studies reported with human fibrins and human plasma, which clearly demonstrated incorporation of dansylcadaverine into both a and y subunits with concurrent inhibition of crosslinking to a polymers and y dimers (28). Incorporation of dansylcadaverine into lamprey fibrin y subunits was Ca"-and thrombin-dependent. These results are in accord with previous reports which demonstrated that with lamprey fibrin as substrate, intrinsic Factor XI11 mediated only the cross-linking of y subunits to y dimers with little or no cross-linking of a subunits to higher polymers (2, 8). Both incorporation of dansylcadaverine into y subunits or cross-linking of y subunits to dimers could be inhibited by iodoacetamide.

DISCUSSION
The problem confronting us now is 2-fold. What are the correct molecular weights of the individual chains and what is the structure of the intact molecule? The quality of the fibrinogen and its constituent subunits that we have been able Experimentally determined apparent partial specific volume. Calculated using * Calculated using GCdc. The number of determinations is given in parentheses.
e Literature cited value. 'Determined as needed to d v e the correct molecular weight for the intact fibrinogen.
--Literature value in aqueous solution.
to utilize in these studies has been such that we have been able to consider these questions from a point of view where that quality was not a limiting consideration. Let us first consider the conventional structure of [(A)az, (B)P2, y~] . If we use the apparent partial specific volumes for the subunits in 6 M guanidine buffer as calculated by the method of Lee and Timasheff (22), then the molecular weights of 78,600, 57,700, and 50,100 for the (A)a, (B)P, and y subunits, respectively, add up to give a presumed molecular weight of 372,300 for the intact molecule in 6 M guanidine buffer. This must be compared to the measured molecular weight of 273,000 for intact lamprey fibrinogen in 6 M guanidine, also obtained using a value for the apparent partial specific volume in 6 M guanidine calculated by the method of Lee and Timasheff (22). While it might be argued that the value of 372,300 is not in bad agreement with the measured values of 352,000 and 358,000 obtained in citrate buffer and the value of 354,500 obtained in guanidine buffer, acceptance of this agreement requires accepting assumptions which do not appear to be warranted. The first of these is that the method of Lee and Timasheff (22) gives values for the calculated apparent partial specific volumes in 6 M guanidine which are appropriate for the individual subunits but not for the intact molecule, or, conversely, that the intact molecule exhibits a marked anomaly with respect to its experimentally determined apparent partial specific volume in 6 M guanidine, but the constituent subunits do not exhibit any anomaly at all. The other assumption is that the gel filtration and gel electrophoresis experiments measured the molecular size of y subunits correctly, but were grossly in error with respect to the (A)a and (B)P subunits.
Due to the fact that the subunits are not soluble in a volatile buffer, it was not possible to obtain their dry weights and, thus, their individual extinction coefficients. Since their exact concentrations in the 6 M guanidine buffer could not be determined, it was not possible to obtain their individual apparent partial specific volumes by direct measurement. Since it appears reasonable that the constituent subunits exhibit anomalous behavior with respect to apparent partial specific volume similar to that exhibited by the intact fibrinogen molecule, the molecular weights of 104,700, 71,600, and 54,300 obtained by sedimentation equilibrium (see Table 111) for the (A)a, (B)P, and y subunits are essentially correct. To the extent that the apparent partial specific volumes of the individual chains differ from that of the whole molecule, these values will exhibit some limited error, but it must be noted that they are in substantial agreement with the values obtained by gel fitration and gel electrophoresis.
Acceptance of these values does require that we give serious consideration to an alternative structure for lamprey fibrinogen, i.e. [ gives a molecular weight of 102,000, corresponding to a molecular weight of 51,000 per y (See Table 11).
The calculation for the partial specific volume of the proteins in 6 M guanidine buffer by the method of Lee and Timasheff (22) is based upon the assumption that there is no significant volume change in the presence of the guanidine and that the amount of solvent components bound to the protein can be calculated. If the actual amount of guanidine bound is less or the hydration is greater than that which is assumed, the calculated partial specific volume will be too small. Calculation of the preferential interaction of component 3, guanidine hydrochloride, using the equation
Here is the calculated partial specific volume of the protein (0.706 cm3/g), I #& is the measured apparent partial specific volume (0.735 cm3/g), p o is the density of 6 M guanidine (1.1418 g/cm3), and C? is the partial specific volume of guanidine (0.763 cm3/g). This negative value may be interpreted either as preferential exclusion of guanidine from the immediate vicinity of the protein or as preferential hydration. Additionally, the possibility of a volume increase must be considered as a possible explanation for the observed increase in apparent partial specific volume.
It may be argued that the intact fibrinogen in 6 M guanidine has a significant amount of structure, that the isolated chains are probably in a random coil configuration, and that the lower calculated values for the apparent partial specific volumes of the isolated chains are more appropriate to this configuration, and, hence, the lower molecular weights for the isolated subunits are also more appropriate. This argument is countered by the molecular sizes obtained for the subunits by gel fitration in 6 M guanidine. It should be noted that the values of 131,000 * 13,600, 65,000 k 3,200, and 53,000 f 2,800 for the (A)a, (B)P, and y subunits, respectively, obtained by this method are in relatively good agreement with the values of 104,000, 71,600, and 54,000 obtained ultracentrifugally. If the gel filtration values are too high, the most probable cause would be that the subunits were not in a random coil configuration. If this is the case, then the subunits going from a more ordered structure in the intact molecule to random coils when isolated cannot be proposed as a cause for a decrease in the value of the apparent partial specific volume of the subunits. Conversely, if the subunits are in a random coil configuration, the agreement of the ultracentrifugal values and the gel filtration values argues that the apparent partial specific volumes are not significantly affected by a structural change. The validity of both sets of values is supported by the values obtained by gel electrophoresis where sodium dodecyl sulfate is the denaturant and by the molecular weight values from mass ratios.
Considering the fact that the apparent partial specific volumes of the subunits are probably not equal and considering the molecular sues measured by gel filtration, gel electrophoresis, and mass ratios, we propose molecular weights of 11O, OOO, 72,000, and 50,000 with a structure of [(A),a, (B)& y2] giving a molecular weight of 354,000. We feel that this is strongly supported by the experimental evidence and by the most reasonable set of assumptions. Considering that the molecular weight and the sedimentation coefficient of lamprey fibrinogen are essentially comparable to those of other fibrinogens strongly suggests that the structure of lamprey fibrinogen closely resembles that of other fibrinogens. Takenvin conjunction with the large molecular weight of the (A),a subunit, we feel that this subunit is probably either a single symmetrically folded chain or is composed of two chains covalently bound by nondisulfide bonds. If the latter is the case, the phenomenon may have occurred during postribosomal assembly and processing of the fibrinogen molecule. We use the subscript n to suggest the possibility of more than one A-peptide per subunit. Correspondingly, the (B)P and y subunits would appear to be single chains. Either of these models appears to be consistent with all of our data and with the generally accepted structure of fibrinogens as a class.
It is now appropriate to consider how the cross-linking experiments and the dansylcadaverine incorporation data may be related to the postulated structure. There is virtually no a polymer formation when cross-linking lamprey fibrin via thrombin-activated intrinsic Factor XIII.
Concomitantly, there is no dansylcadaverine incorporation into the a subunit or into the traces of a polymer. These results are obtained with either lamprey or human thrombin. It should be noted that only the lamprey thrombin removes the lamprey Apeptide, the human thrombin not being effective in this regard although both cause the removal of the B-peptide(s). In contrast to the unusual behavior of the a subunit, the lamprey y chains rapidly form dimers under the influence of calciumdependent thrombin-activated intrinsic lamprey Factor XIII. Dansylcadaverine is incorporated into the y chains and to a limited extent into the y dimers. Thus, in this latter respect, and with respect to the fact that B-peptides are released, lamprey fibrinogen behaves in a manner analogous to all previously studied fibrinogens.
It is possible that cross-linking of lamprey fibrin may be regulated by the level of Factor XI11 present in the plasma. Indeed, efforts to isolate lamprey Factor XI11 from a large quantity of plasma were unsuccessful. The conventional Factor XI11 assay using high specific activity 3H-labeled putrescine (29) under various conditions showed negligible activity in whole plasma as well as in concentrated fibrinogen solutions. However, from the cross-linking patterns observed on polyacrylamide gels, it is evident that a Factor XIII-like enzyme participates in stabilizing lamprey fibrin clots.3 It is unlikely that the a subunit does not cross-link because there are no cross-linking sites available to lamprey Factor XIII, since in previously reported experiments, lamprey a subunits could be cross-linked by the addition of human Factor XI11 (2,lO). Lamprey Factor XI11 is activated by either human or lamprey thrombin and is Ca2+-dependent. It is also inhibited by sulfhydryl reagents.
It is interesting to note that thermal stability of clottable proteins reflects the animals adaptation to its environment. Lampreys, which inhabit a cold environment, fall into this category, as does the Pacific salmon. Lamprey fibrinogen exhibits the lowest inherent stability of any of the vertebrate fibrinogens in terms of thermal denaturation, denaturing a t 31°C as compared to salmon denaturing at 45"C4 and mammalian fibrinogens denaturing at 56°C. Thus, since lamprey fibrinogen differs significantly from other fibrinogens only with respect to the (A)a subunit and not with respect to the (B)P and y chains, this lower thermal stability may be yet another reflection of this unusual ~tructure.~ This fibrinogen comes from one of the most primative vertebrates and possesses certain properties which are distinct from those of the rest of the species examined in detail thus far. Nevertheless, this protein still performs the same function in the lamprey as its counterparts in higher vertebrates. In collaboration with Dr. J. W. Donovan, United States Department of Agriculture, Western Regional Research Laboratory, Berkeley, California, we have examined a number of our preparations of lamprey fibrinogen by differential scanning calorimetry. The protein exhibits two endotherms similar to a pattern reported from native bovine fibrinogen or a mixture of D and E fragments of the same molecule (30). However, the endotherm for the lamprey D domain appears at a lower temperature, 5OoC, compared to 60°C for bovine. The endotherm for the E domain appears at a higher temperature, 105OC, compared to 100°C for bovine. The results would indicate that this primitive fibrinogen possesses regions of ordered structure that have remained relatively unchanged throughout evolution.