Nov., 1947
DOUBLE REFRACTION OF FLOWOF HUMANFIBRINOGEN AND PLASMA FRACTION I 2731
fibrinogen concentration of about 0.1 g./liter, a PH of about '7.3, and a relatively high thrombin concentration, both incomplete precipitation of fibrinogen and occlusion of other proteins probably occur. On the basis of this study, values for the fibrinogen content of normal human plasma in the literature may well be too high. In view or"the very high occlusion of the lipoproteins, Fractions IV-1 and 111-0, reports of elevated fibrinogen levels in pathological conditions involving concomitant increases in lipoprotein should be viewed with some circumspection. Recent reports2*of a. protein of high molecular weight and considerable asymmetry, which may occur in concentrations up to 50-7070 in pathological plasma are pertinent in this connection. At such concentrations a protein with an occlusion factor of only 10 would cause apparent yields 2 to :3 times greater than the actual fibrinogen content. Acknowledgments-We are indebted t o Miss Susan G. Miller for her assistance in many of these analyses and to Drs. E. J. Cohn, J. T. Edsall, and J. D. Ferry for their generous advice.
Summary 1. Analytical procedures for fibrinogen are briefly summarized and several convenient modifications of technique are described. 2 . The yield of fibrin obtained by the action of human thrombin on human fibrinogen, and its modification under various conditions of p H and ( 2 8 ~( a i K. 0. Pedersen, "Ultracentrifugal Studies on Serum and Serum Fraction," Uppsala. l ! l . i 5 p 38, 121. (b) J. WaldenstrBm, in " T h e bvedherg, 1884 :iOjS 1944,'' Lppsala, 1814, p. 538-573.
concentration, and by the presence of other proteins, has been studied. 3. At a pH of 6.3 complete yields were obtained a t fibrinogen concentrations between 0.5 and 2.0 g./liter and at thrombin concentratiops between 0.05 and 0.20 unit/cc. Reduced yields were obtained at both higher and lower concentrations of both reactants. 4. Other proteins were carried down by fibrin in amounts which varied widely with their nature. These ranged from a negligible occlusion of serum albumin to a 10-%70 occlusion of certain lipoproteins and an almost complete occlusion of several enzymes, including thrombin. In general, the occlusion of each added protein was proportional to its concentration. Certain plasma components were shown to be occluded in amounts sufficient to introduce a considerable error into analyses of plasma fibrinogen. 5 . A study of the effect of the fibrinogen and thrombin concentration and of the pH on occlusion revealed t h a t in each case the condition which favored occlusion also favored the formation of a "fine" clot. This, and the fact t h a t proteins of high molecular weight and asymmetry are strongly occluded, while smaller, more asymmetrical ones are not, indicates t h a t physical entrapping is important, although the polar and nonpolar interactions of the molecules may also play a part. 6 . Optimal conditions for complete yields and minimal occlusion are defined, and a procedure for estimating the extent of occlusion is described. RECEIVED MARCH8, 1917
BOSTON, XIASS.
[ C O S T R I l i L T I O X FROM THE DEPARTMENT OF PHYSICAL CHEMISTRY,
HARVARD MEDICAL
SCHOOL]
Studies on Double Refraction of Flow. 111. Human Fibrinogen and Fraction I of Human Plasma' 131' J O I I N
T.
EDS.\LL, JOSEPII
F. I;OSTER?
Fibrinogen, as the major constituent involved i n the clotting o f blood, is a protein of particular
chemical and biological interest. In the past, however, its instability has rendered difficult the study of its physico-chemical properties. The large-scale fractioiiation o f blood plasma, employing ethanol a t low temperature and low ionic ( I I 'l'hi, jralwr is Suin1,cr 1, 1 in t h e series "Stn T h e high intrinsic viscosity of fibrinogen i n solut i o i l , " an(l thc re:itliness with which it is convertetl by thrombin into the complex network of the 1il)riri clot, inclicate that the molecule is eloilgateti J.
( : 3 ) I,,.J. Cilhri, I. I:. Strong, M', I,, I I u g I i e ~ .J r . . I ) . I . l l i i l l < ~ r d , S :l\h\\- of them. The relative inaccessibility of serine prompted a search for a convenient and economical synthesis that would give a good yield of product without the use of high-pressure amination or drastic acid hydrolysis of an ether linkage. The present paper reports such a synthesis. In the Knoevenagel condensation of formaldehyde with ethyl malonate in the presence of catalytic amounts of diethylamine the product iso-
uct, methylolmalonic ester, is the initial reaction product. Apparently, however, this aldol-like pri inary condensation product has never been isolated from the reaction because of its tendency to undergo dehydration to methylene malonic ester or further condensation to the bis compound. Should formaldehyde similarly condense with the now readily available ethyl acetamidomalonate17*i8,19 the product, ethyl a-acetamido-a-carbethoxy-,@-hydroxypropionate (I), would be incapable of intramolecular dehydration and might be expected to produce serine (11) on acid hydrolHC(COOCzH5)z f HCHO
I
IGHCOCHX HOCHzC(C0OCzHs)z --+ HOCHzCHCOOH
I
I
NHCOCHi
NH2
I
I1
ysis. Formaldehyde and ethyl acetamidomalonate were found to condense to give a quantitative yield of I. However, concentrated hydrochloric acid hydrolysis of I caused complete destruction of the molecule and gave no serine; the nitrogen came out as ammonium chloride and the rest HCHO $. CHz(C0OCzHr)z + [HOCHzCH(COOC~Hs)zI of the molecule was converted to pyruvic acid. The same results were obtained with 1 N hvdrochloric acid. This behavior appears more rational after lated is the bis compound, ethyl a,a'-tficarbeth- consideration of the electronic structure of the o x y g l ~ t a r a t e , 'but ~ when a trace of caustic soda molecule. The oxygen of the hydroxyl group in is used as the condensation catalyst it .has been the molecule is inherently nucleophilic and, in the that the bimolecular condensation prod- presence of strong mineral acid, is susceptible to proton attack, forming the oxonium ion A. Under (1) Presented before t h e Division of Organic Chemistry of t h e American Chemical Society a t t h e 111th Meeting, Atlantic City, the reaction conditions employed the ester linkApril 14, 1947. (2) Present address: Warner Institute for Therapeutic Research, 113 West 19th Street, New York, N . Y. :3j Fiscller and Leuchs, Bey.. 56, 3788 (1902). ( 4 ) Lruchs a n d Geiger, ibid., 39, 2644 (1906). ( 5 ) Dunrr, Redemann a n d Smith, J. Biol. Chem., 104, 511 (1'334). ( 6 ) Redemann a n d Icke, J . Org. Chem., 6, 1.59 (1943). (7) Erlenmeyer, Ber., 36, 3769 (1902). (8) Erlenmeyer a n d Stoop, Ann., 357, 236 (1904). (9) M i t r a , J . I n d i a n Chem. Soc., 7 , 799 (1930). (IO) Maeda, Terumi and Suzuki, Bull. Znsl. Phys. Chem. Research (Tokyo). 1Y, 267 (1938); C. A , , 34, 6931 (1940). (11) Schiltz and Carter, J. B i d . Chem., 116, 793 (1936). (12) Carter a n d West, Organic Syntheses, 20, 81 (1940). (13) Wood a n d d u Vigneaud, J. Biol. Chem., 194, 413 (1940). (14) Mattocks a n d Hartung, i b i d . , 166, 501 (1946). (15) Knoevenagel, Ber., 27, 2346 (1894). (16) D'Alelio, U. S. P a t e n t 2,330,033; c j . Welch, J . Chem. Soc., 257 (1930), who believes t h a t the primary reaction product is monomethylolmalonic ester when amine catalysts are used.
COOC~HI
I
H I
H-O-CH~-C-COOC~H~
I
@
NHCOCH,
A
o=c-oe I;I
PI
H-A-cH~-c--COOH 1'
@
+ HB
I
NHCOCHI B
(17) Locquin, Bull. soc. chim., [4] 49, 42 (1931). (18) Snyder and Smith, THISJOURNAL, 66, 350 (1944). (19) Albertson, Archer a n d SGter, ibid., 66, 500 (1944); 67, 38 (1945).
