Physicochemical studies of fractionated bovine heparin

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Physicochemical

Studies

III. Some Physical S. S. STIVALA, Department

122, 32-39 (1967)

of Fractionated

Parameters L. YUAN,

of Chemistry Received

in Relation

J. EHRLICH;

Bovine

Heparin

to Biological

Activity1

AND

P. A. LIBERT13

and Chemical Engineering, Stevens Institute Hoboken, New Jersey 07030 March

27, 1967; accepted

April

of Technology,

21, 1967

Graded self-hydrolysis of commercial bovine heparin produced samples of varying anticoagulant activity. The hydrolysis results in desulfation, which is accompanied by decrease in (a) sedimentation coefficient, (b) intrinsic viscosity when measured in water, and (c) axial ratios, a/b, if a prolate ellipsoid model is assumed. Intrinsic viscosities obtained in 0.5 M NaCl were relatively unchanged for the hydrolyzed samples. The drop in molecular weight during inactivation is due to desulfation and not to depolymerization. Hydrolysis result,s in both 0- and N-desulfation, the latter to a greater extent than the former. The dat,a suggest that degree of sulfation and size and/or shape confer on heparin its biological activity.

It has been suggested that anticoagulant activity of heparin is related to (a) structural features, such as sulfation (11-13) and carboxylat’ion (13), and (b) molecular shape and size (11, 13, 14). These factors are related to anticoagulant activity by virtue of their importance in the binding capacity of heparin (13). This paper is a preliminary present,ation of an investigation undertaken to examine various structural features, including size and shape, of heparin in relation to anticoagulant activity. Additional parameters will be reported in subsequent papers.

The widely distributed, sulfated mucopolysaccharide heparin, found in high concentration in liver, lung, and spleen, is important in the clearing reaction and pharmacologically as a blood anticoagulant. Although the biological activity of heparin is believed to be attributed to structural features as well as physical parameters, the basis of anticoagulant activity has not been fully established. Heparin is heterogeneous in molecular weight (l-6), with fractions varying from approximat’ely 6000 to 20,000. Because of its high negative charge, heparin behaves in solution as a typical polyelectrolyte (6-9), and t,hus makes measurements based on hydrodynamic or osmotic properties particularly sensitive to environmental conditions of ionic strength (5, lo), pH, and concentration.

MATERIALS

AND

METHODS

Pure commercial heparin sodium samples, derived from beef lungs, were obtained from Organon, Inc., West Orange, N. J.; Lot No. 22419 assayed at 157 IU/mg. All measru-ements were made on unfractionated heparin dried over PzO5 at 25” for a minimum of 2 days. Ilt-120 (20-50 mesh, Mallinckrodt) was converted from the sodium to the hydrogen form and boiled in several changes of distilled water. Potassium hydroxide and KC1 were used to repeatedly convert IRA-400 from the chloride to the hydroxyl form. This resin, after washing on a column with

1 This work was supported by grant HE-05943 from the U.S. Public Health Service. 2 U.S. Public Health Service Fellow (5F2-HE29038). 3 Present address: Department of Biochemistry, Jefferson Medical College, Philadelphia, Pennsylvania 32

FRACTIONATED distilled water to remove excess hydroxyl ion, was employed in the hydroxyl form. Approximately 0.1 N solutions of carbonate-free NaOH were prepared in freshly boiled water and standardized against potassium acid phthalate. Inactivation of hgparin by hyclrolysis. The inact,ivation of heparin into simples of varying anticoagldant activities was achieved by self-hydrolysis. The hydrolysis was accomplished by a modification of a procedure described by Helbert and >Iarini (15). iin accllrstely weighted quantity of heparin sodium (7.8500 gm) was dissolved in several milliliters of distilled water. The sodium heparinate solution, kept at O”, was converted to heparinic acid by slowly percolating it through a column of the IR-120 in the hydrogen form (the quantity of resin employed was about five times the estimated amount necessary). The column was subsequently flushed with ice-cold distilled water, and the effluent was collected in a l-liter volumetric flask until no heparin was detected with toluidine blue; yield was heparinic acid of concentration of approximately 1 gm/lOO ml. One hundred milliliters was removed as a “zero” time aliquot and immediately refrigerated at 5”. The remaining solution in the flask was placed in a constant temperature bat,h at 57 + 0.5” to effect graded hydrolysis for various periods of time. Aliquots of 100 ml were removed every 135 hours over a t,otal of 6 hours, including aliquots following 24 and 72 hours of hydrolysis. The react,ion was stopped by rapidly cooling each aliquot in nn ice bath immediately after removal from the reaction bath (57”). Each s.iiquot was divided into two equal volumes and treated as follows: (a) 50 ml was first titrated with 0.1 N NaOH to pH = 12, backtitrated with 0.1 N HCI to the equivalence point, and then passed through a column of IRA-400 (in the hydroxyl form) to remove sulfate radicals from hydrolysis; (b) the other 50 ml was first passed through the column of IRA-400 to remove hydrolyzed sulfate radicals, then titrated with 0.1 N XaOH to pH = 12, and back-titrated with 0.1 N HCI to the eqllivalence point. The two 50 ml of parts (a) and (1)) of each aliquot were then combined and the SaCl (resulting from the backtitration) was removed by reducing the volume by evaporation; the heparin was then precipitated with cold absolute ethanol. After overnight storage in a refrigerator, the supernatant fluid was removed by centrifllgation at 15”. Samples were reprecipitated twice, lyophilized, and stored over P:Os for a minimum of 48 hours prior to study. Tot,al desulfation for each aliqnot (resulting from t,he graded hydrolysis) was calculated bysubtracting the number of titratable groups in (a)

BOVINE

HEPARIN

33

from those in (b). Calculations of 0-desulfation and N-desulfation are based on earlier work by Helbert and Marini (15). Chemical analysis and bioassay. Sulfur analysis was performed by Schwarzkopf Microanalytical Laboratory, Woodside, New York. Anticoagulant activity was determined according to the USP XVI method by the South Mountain Laboratories, h/laplewood, New Jersey. Titration. A Beckman Zeromatic pH meter with glass electrodes and type K-100 calomel reference was employed. A temperature-controlled bath was used so that specimens were thermostatted at 25 f 0.1” prior to titration. The pH meter was standardized at pH 4, 7, and 12 against standard buffer solutions (Beckman). Viscosity. \‘iscosity measurements were performed in a semimicro Cannon-Ubbelohde dilution viscometer. Because of the long efflux time (between 206 and 800 seconds) and low molecular weight of the fractions, kinetic energy and shear corrections were not applied. The flow times at 25.000 f 0.003” for solvent and solution were determined to within 0.1 second with a stopwatch. Determinations were made using partially hydrolyzed samples dissolved in water and in NaCl (Baker analyzed reagent grade) solutions using filtered deionized distilled water. All heparin solutions were introduced into t,he viscometer after filtration through a medium fritted disc. Serial dilutions were made in the viscometer. Sedimentation equilibrium. A 1% solution of the heparin aliquot was prepared by dissolving milligram quantities in 20 ml of 0.5 M sodium chloride (Baker analyzed reagent grade). The solutions were dialyzed at 5” in prewashed Visking sausage casing sacks against solvent with continuous magnetic stirring for 48 hours. The dialyzate was used as solvent for all dilutions. The pH of all solutions was found to be approximately 6 after dialysis. An analytical Beckman-Spinco model E ultracentrifuge with a four place An-G rotor, epoxy centerpieces, and wedge windows was used in equilibrium studies. Rayleigh interference optics were employed. Precision volume measurements were made with Hamilton microliter syringes equipped with Chaney adapters. Fluorocarbon (Union Carbide FC-43) was Ilsed in both chambers of the cells. Column lengths of solutions varied from 1.5 to 2.5 mm. Runs were made from 18 to 24 hours at 15,220 rpm. Temperature was maintained at 20” with the RTIC system. Initial concentrations were determined in the ultracentrifuge with a Kegeles double sector synthetic boundary cell in an Arr-D rotor at 5220 rpm. Photographs were made on Kodak Type 11-G spectroscopic plates. Measurements were made with the Nikon Shadow-

34

STIVALA,

ET AL.

TABLE SOME Time of hydrolysis (hours)

Sample

AB-0” D-lb D-l D-2 D-3 D-5 D-6

PAR~IMETERS

I

RELATION

IN

TO BIOLOGICAL

ACTIVITY

A;$ti? units/mg)

148 128 99 56 26 0 0

0 1.5 3.0 4.5 24 72

Q Original b Original

PHYSICAL

unfractionated unfractionated

13,100 12,700 12,400 11,700 11,400 11,100 10,700

12,900 12,750 12,670 11,600 11,400

11,200

sample. sample immediately

after

2.44 2.36 2.22 2.27 2.14 1.81 1.61

passing

0.170 0.182

5.10 4.00 3.45

0.37 0.37 0.40

135.5 122.5 114.0

0.186 0.188 0.177

3.37 2.6 2.0

0.40 0.50 0.50

112.5 96.5 82.5

through

In-120

in the hydrogen

form,

T = 0". c Calculated for the Na salt from known amount, of desulfation (titration data) and original weight (13,100). d Calculated for the Na salt from known amount of desulfation (S analysis) and original weight. e Determined in 0.5 M NaCl from sedimentation equilibrium. graph model 6 comparator. Molecular were calculated from the equation

2RT Mw

=

(1

-

J7p)wz

cb . C,(XbZ

-

weights

c, (1) -

X,2)

’

where Co is the initial concentration of solute, Ch and C, are concentrations at the cell bottom and meniscus, respectively, and Xb and X, are the distances from the center of rotation of the cell bottom and the meniscus. p is the part,ial specific volume, taken as 0.470 for heparin (5), p is the solution density determined by pycnometry, and w is the angular velocity in radians per second. Six concentrations were employed for each determination. Since the apparent molecular weight of heparin is concentration dependent, the recip.. rocals of the apparent mclecular weights were plotted against concentration, and an extrapolation to zero concentration was made by least squares. The inverse then is the molecular weight. Seu’im:ntation velotity. Sedimentation velocity measurements w-ere made at 20 f 0.02”, in 0.5 M NaCl solution. Capillary-type synthetic bolmdary cells were ut,ilized in an AN-D rotor at 59,780 rpm. Schlierin photographs were taken at %minut,e intervals. Since the patterns were symmet,rical, measurements were made from the reference line to the maximum ordinate. As the apparent sedimeutation coefficient of heparin js concentration dependent, an extrapolation of the reciprocals of the apparent sedimentation to zero concentration was made. At least five different concentrations for each determination were used. The calculations were done on an IBM 1620 computer with a program (30) in Fortran II.

molecular molecular

RESULTS

Table I summarizes some measured and calculated physical paramet’ers, and Table II contains chemical groups and sulfur analysis of heparin, in relation to biological activity. The change in anticoagulant1 activity of heparin, shown in column 3 of Table I, accompanies time of hydrolysis. Since hydrolysis of heparin results in desulfation, the data contained in Tables I and II would be in terms of desulfation, i.e., degree of sulfation of heparin. Molecular weights. Columns 4, 5, and 6 of Table I show decreasing molecular weight as a function of time of hydrolysis. This is to be expected. Sulfate groups are released during hydrolysis, as indicated in Table II and reported by others (12, 13, 15, 16). Column 4 contains molecular weight, M,‘, calculated from the measured (sedimentation equilibrium) molecular weight of the unhydrolyzed heparin (sample AB-0) and the determined total desulfation from titration data. Column 5 contains molecular weight, M,“, calculated from sulfur analysis (last column of Table II). Column 6 shows two measured molecular weights, MW , one for sample AB-0 and the other for sample D-6, hydrolyzed for a period of 72 hours. M, dropped from 13,100 to 11,200, which is a reflection on the number of sulfate groups released from the

FRACTIONATED

BOVISE TABLE

DESULFATION

IN

RELATION Sulfate

II

TO groups

35

HEPARIS

ANTICOAGULANT

ACTIVITY

releaseda

74s

A(‘:$ty Apparent total desulfation (B)

Sample units/n&

AB-O D-O D-l D-2 D-3 D-5 D-6 o Titratable b Calcnlat~ed

148 128 99 5G 26 0 0

0 0.2 1.4 2.6 4.9 7.7

0 6.0 17.5 21.7 25.9 32.2

&-Desulfation” (0

0 2.9 8.05 9.55 10.5 12.25

Calc’d.

Observed

11.1 10.5 10.1

11.41 11.14 11.42

8.5 8.0 7.0

11.43 9.41 8.75

groups per heparin molecule. as (B-A)/2 (Ref. 15).

chain. During the drol) in molecular weight, due to desulfation, the anticoagulant act)ivity decreased from a high of 148 U/mg to zero activity. The experimental value of 11,200 compares favorably with the calculated values of 10,700 and 11,400. Various invcst,igators (5, l-1, 16) have report’ed litt’le change in molecular weight in inactivating heparin with warm acid. This is not, essenkdly inconsistent with the above since their inactivation studies were not conducted to the extent of 72 hours. As not)ed in Table I, the drop in molecular weight is not appreciable (I 2,700-l 1,400, or 12,900-l 2,670) in going from 118 to 26 U/mg. This drop in molecular weight reflects desulfation. Therefore, t,hc self-hydrolysis does not result in depolymerization, an observation also reported by Jensen and co-workers (14). The observation that [v], measured in O..; s KaCl (column 8), does not al)preciably change, within experimental error, - further demonst&es the constawy of DP (average degree of polymerization) of heparin during the hydrolysis, which was calculated to be 22, i.e., repeat units per chain. Various investigators (3, 5, 6) have reported that biological activity is related t,o molecular weight. However, t’heir reported observations were based 011 bioassays of fract,ions of heparin (fractionat,ed from a single preparation) of varying DP but, having essentially identical chemical composit,ion. On the other hand, the variation in biological act’ivity (column 3, Table I) is not a manifestation of molecular weight change (col-

umns a-6), since DP remains constant,, but rat’her of chemical variation among t’he samples as a result, of desulfation. Sedimentation

coeficient

and intrinsic

&

cosities. Column 7, Table I shows t,hat the sedimentation constant, so, decreases with time of hydrolysis, and in effect, desulfation. This is not unexpected since the sedimentation constant is related to molecular weight. Column 8, Table I contains values of [q] of hydrolyzed heparin samples measured in 0.5 N NaCl. These were obtained by extrapolating to infinite dilution the linear plots of ~sp/c versus c, expressed by the Huggins equation (17) : ?Isplc= Irll + li’hlk.

(2)

As not’ed, there is 110 al)preciable change within experiment’al error, and support is given the premise of 110 depolymerieat,ion during hydrolysis. However, any change in electrostatic interaction due to desulfation is overwhelmingly swamped by the high salt, concentration. Plots of vsp/c versus c of hydrolyzed heparin samples in water were nonlinear, typical of polyelectrolytes. Column 9, Table I, contains the intrinsic viscosit,ies, A, measured in water, calculated from t’he empirical relation developed by Liberhi and Stivala (9) : ?l.qJc =

A 1 + B(c”2

-

kc) ’

(3)

where A and B are constants characteristic of each fract,ion, and k is an interaction

STIV,4LA, &-kc

J

0I

I 0.1

I 0.2

I, 0.3

FIG. 1. Reciprocal cosity versus square sample D-O.

k=0.37

I,, 0.4 JE

0.5

0.6

0.7

,

(

0.6

0.9

of reduced aqueous root of concentrat~iou

visfor

constant for a given polyelectrolyte-solvent system. Equation (3) shows that the intrinsic viscosity, i.e., the limit of &c as the concentration approaches zero, is equal to A. The term B expresses t’he concentration dependence of the reduced viscosity. The aqueous viscosity data were fitted to Eq. (3) by plotting (&c)-l versus c1j2 and adjust’ing k until all curves were linear, as shown in Fig. 1. The value of k required to make the curves linear was found to vary for the different samples. Column 10, Table I shows that Ic varies from 0.37 to 0.50. It was suggested (9) that variation in k reflects chemical heterogeneity among fractions since it is a measure of electrostatic forces between t,he polyion and its counterions. Samples AB-0 to D-6 are indeed chemically different by virtue of their varying degree of sulfation. Hence, their varying electrostatic forces, i.e., solute-solvent interaction, are reflected in the various values of k. Liberti and St)ivala (9) obtained a value of 0.25 for k of all fractions of heparin fractionated from a

ET AL.

single preparation (chemical compositicn essent,ially ident,ical). Column 9, Table I shows that [v] of hydrolyzed heparin samples measured in water decreases with time of inactivation. That this was not exhibited by [q] measured in 0.5 K NaCl clearly demonstrates the swamping effect of the salt, where also small changes in molecular weight are not detected. In the absence of salt, the int,rinsic viscosity becomes more sensitive to variation in electrost’atic interaction. Desulfation results in diminished charge density and causes the chain to become less extended. Hence, decreasing [a] in water accompanies desulfation. This is also demonstrated by decreasing axial ratios, if a prolate ellipsoid is assumed in water for the heparin samples, as shown in last column of Table I. Liberti and Stivala (6), using the Simha relation (18) for prolate ellipsoids, calculated axial ratios of heparin fractions (fractionated from a single preparat’ion) from [q] in water and molecular weights (determined from sedimentation equilibrium data). The values of axial ratios, a/D, in Table I are approximate since they were obtained from dat,a reported by Liberti and Stivala (6) on a/b, [v], and M, . Nevertheless, the a/b data shown in Table I serve to demonst)rate the changing tendency in this parameter with inactivation, or desulfation. Table II contains desulfation data for the samples, as a function of activity ior time of hydrolysis). Apparent total desulfation, N-desulfation, and 0-desulfation were ralculated from titration data. It is noted from columns 3 and 5, Table II, that N-desulfation occurs at a much faster rate than does 0-desulfation, which is consistent with that reported by Helbert and Marini. Thus, the self-hydrolysis involves rapid cleavage of Nsulfate groups followed by slower release of the O-sulfate groups. Various investigators (19, 20) have shown that acid hydrolysis of heparin under certain conditions (0.04 N HCl at 95-100” for about 2 hours) can result in loss of all the N- and only some of the O-sulfate groups. The labile nature of Nsulfate groups to acid has also been confirmed through the use of model compounds (21, 22).

FllA~‘TTOSATED

The last two columns of Table II contain the percentage of sulfur that was calculated (from titration data, and molecular weight of original unhydrolyzed heparin) and observed (analysis). The decrease in sulfur parallels time of hydrolysis, and the agreement between observed and calculated is quite good. It is noted that’ desulfation accompanies loss of activity. This observat,ion has been reported (I l-13, 16). Table II also shows that there is about 50 % loss of N-sulfate compared with about 17 % release of O-sulfate in going from 130 to zero CSP units/mg of activity. Plots of biological activit#y against’ 0- and ,V-desulfation showed that’ a modest drop in n&v&y is accompanied by a small reduction in Osulfa& and a relatively sharp drop in Nsulfates. DISCUSSION

It has been suggested (1) that the high anticoagulant activity of heparin is due to t,he concerted effect of a series of molecular features which include (a) degree of sulfat,ion and (b) molecular size and shape. Data recorded in Tables I and II support the above. For example, the data show that’ the selfhydrolysis of heparin is accompanied by inactivation which in t’urn results in desulfation, decrease in aqueous viscosity, decrease of axial ratios a/b for prolate ellipsoid model in water, and decrease in sedimentatjion coefficient . Degree of suljution. The contjribution of sulfate groups of heparin t#o anticoagulant activity has been well demonstrated, t,hough this by no means is the sole consideration. Deactivation studies in t,his investigation, and those conducted by ot’hers (12, 13, 16, 23), show the relationship between anticoagulant, activity and degree of sulfation. Yet’, further sulfation (16, 24) (from 12 to l-1.4 %) of a highly act,ive sodium heparinate resulted in a decrease in activit,y of more than 50% rather than an increase. Further, many sulfated polysaccharides (24, 25) having a sulfat,e content higher than heparin exhibit,ed anticoagulant art,ivit,y considerably below that of heparin. Even dextran (26), sulfated to the same degree and greater than heparin, showed anticoagulant activity

BO\-IKE

HEPARIS

37

much below t,hat of heparin. Thus it) is evident that degree of sulfation of itself is not, sufficient in rendering heparin its biological property. Recently, Stivala and Liberti (13) reported on the importance of the carboxylic groups of heparin in relation to activity, and to divalent, cation binding. It would appear from t,he data in Table II that N-sulfate groups in heparin are more vit#ally associat,ed with anticoagulant activity than the O-sulfates, since more of the former are released during hydrolysis. However, the reverse might’ have prevailed were the ester sulfates more labile than t’he sulfoamino groups. McAllister and Dcmis (27) studied the metabolism of exogenously administered heparin in man. They reported t,hat uroheparin, the principal early metabolite of heparin, appears t’o be a mucopolysaccharide of composition similar t,o heparin, formed directly by the removal of an Osulfate (ester sulfate) from each repeating unit. They found that t)he anticoagulant act.ivity of uroheparin is less than heparin. It is interesting to note t’hat select,ive iVsulfation of chitosan, using t#he pyridinesulfur trioxide complex in aqueous alkaline medium, yielded (28) a product which exhibited no anticoagulant activity. On t#he other hand, treatment of t,his material wit,h sulfur dioxide and sulfur trioxide produced an N-sulfated, 0-sulfat’ed (~7.5 70) chitosan having considerable activity. Coleman and co-workers (29) also sulfated chitosan with SO2 and SOS and obtained products of low activity. It may t,hus be that anticoagulant activit’y is not contributed by a particular type of sulfate group but rat,her perhaps in combination with the carboxyl groups (13) and t,he overall shape and/or size of the chain. :l/olecular parameters. The drop in molecular weight wit’h inactivat,ion (Table I) is due to desulfation and not depolymerization. Hence, any correlation made bet,ween molecular weight and activit,y in this case would not relate the effect of chain length. However, various invest)igators (3, 5, 6) have reported on the relationship between degree of polymerization (molecular weight) and anticoagulant activit,y. For example, Laurent (3) reported that biological activity

38

STIVALA,

increased linearly with molecular weight for fractions of a single preparation of heparin, and Stivala and co-workers (5, 6) showed that anticoagulant activity initially increases with increasing molecular weight and levels off beyond some value of molecular weight. Thus the anticoagulant activity (A) increases with molecular weight (M) in accordance with the empirical equation (Ref. 13) 1/A

= a + b/M,

(4)

where a and b are constants. The drop in (a) [q] measured in Hz0 (5.10-2.0 dl/gm), (b) sedimentation coefficient, so (2.44 X lo-l3 to 1.61 X lo-13), and (c) axial ratios, a/D (135.5~82.5), shown in Table I, indicate size and/or shape changes with inactivation. The changes reflect the changing electrostatic interactions of the heparin samples as a result of desulfation. Since desulfation parallels inactivation, the above physical parameters appear to be related to activity. In the course of repeated recrystallization of heparin in warm acetic acid, Jensen et al. (14) found a considerable drop in the anticoagulant activity of the original heparin. They reported that as the activity fell (10044%) there was (a) an increase in the sedimentation rate (~20 = 2.07 to 2.7), (b) no appreciable change in molecular weight (17,000-16,600 as determined from the diffusion constant), (c) no decrease in the sulfate content, and (d) a drop in the frictional ratio from 2.5 to 1.81. It was suggested that a “structural rearrangement” had occurred, such as a change in shape or hydration. Though the data reported in this paper are not quite consistent with those of these investigators, yet their work along with data contained in Table I substantiate the hypotheses of some size and/or shape change during inactivation. Wolfrom and co-workers (16), on the other hand, reported that the inactivation of the sodium salt of heparin with phosphate buffer at pH 2.6 at 25” for 48 hours was accompanied by (a) a decrease of sulfur content from 12.9 to 8.5 %, (b) elimination of amino groups, (c) an increase in optical rotation, and (d) no change in relative vis-

ET AL.

cosity. Data in Table I show no change in [g] when obtained in NaCl solution. Thus the suggestion that the high anticoagulant activity of heparin is due to the concerted effect of a series of molecular features which include (a) degree of sulfation and (b) molecular size and shape, appear established. However, the contribution of the carboxyl groups may also be important (13). However, how the above features contribute has not been fully elucidated nor reported. Recently, however, Stivala and Liberti (13) reported that these features confer on heparin its anticoagulant activity by virtue of their role in the binding process. Their work was based on Cu(I1) binding of heparin in relation to pH, molecular weight, and biological activity. Desulfation and biological activity parallel Cu(I1) binding of heparin. ACKEOWLEDGMENT The authors are grateful to Organon, their generous sl~pply of heparin sodium.

Inc. for

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FRACTIONATED 14. JENSEN, I:., SNELLMAS, O., .~ND SYLVAN, B., J. Biol. Chem. 265 (1948). 1.5. ~IELDERT, J. I<., AND ~IARINI, RI. 4., Hiochenistrg a, 1101 (1963). 16. WOLFRAM, AI. L., AND RIc.~YEELB, W. H., J. Am. Chem. sot. 67, 718 (1945). 17. HUGGINS, 11. L., J. -lm. (‘hem. Sot. 64, 2716 (1942). 18. SIMHz4, Ii., J. E’h!/s. Chew. 44, 25 (1940). 19. Jowes, J. E., BOSTR~M, II., ANI) ~IUTT, I'., J. Hiol. (‘hem. 183, 607 (1950). 20. FOS,rER, A. B., 1\1AH'I'LEW, E. F., AXL) S'I'ACEY, hl., Cham. Intl. 899 (1953). 21. FOSTER, A. B., ~IARTLEXV, E. F., S,rac~y, ill., TAYLOR, 1'. J. M., AND WEBBER, J. M., J. (“hem. Sot. 1201 (1961). 22. (;IIJI~NS, I(. A., AND WOLFROM, $1. L., dxh. Niochun

J?iophys.

98, 371 (1962).

BOVINE

HEPARIN

39

23. KOBAYASHI, Y., .Irch. Biochem. Hiophys. 96, 20 (1962). 24. WOLFROM, M.L., SHEN, T. M.,ANI) SUMMERS, C. G., J. 4m. (‘hem. Sot. 75, 1519 (1953). 25. MEYER, K. II., PIROUE, l<. P., AND ODJER, RI. E., Helv. Chine. ilcta 35, 574 (1952). 26. BICICETTS, C. K., Biochrm. J. 51, 129 (1952). 27. MCALLISTER, B. &I., AND DEMIS, U. J.,!Vature 212, 293 (1966). 28. WARNER, D. T., AND COLEMAN, L. L., J. Org. Chem. 23, 1133 (1958). 29. COLEMAN, L. I,., XICCAIUY, L. I',, WARNER, D. T., WILI,Y, 1:. F., 4~1) FLOKRTRA, J. I-I., Abstr. Papers .4m. (‘hem. Sot. 123, 19L (1953); (Ref. 11). 30. EHRLICH, J., WEIXER, I'., AND STIVALA, S. S., J. Macromol. Chem. in press.