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BIOCIIIM[CA ET BIOPHYSICA ACTA IO 3
BI~A 25943
H E P A R I N : MOLECULAR WE[GIqT AND DEGRADATION STUD1ES*
EMOI~Y B R A S W E L L
Biochemistry and Biophysics Section, University of Connecticut, Storrs, Conn. (U.S.A.)
(Received September ~-7th, I967)
(Revised mamlscript received December 4th, I967)
SUMMARY
z. Previously published molecular weights of heparin determined by sedimen- tation have been found to be incorrect and are probably too low by about I6 %. By means of equilibrium sedimentation, the study of molecular weight heterogeneity and the determination of both Z- and weight-average molecular weights was made. Heparin was found to be quite heterogeneous with an average ratio of Z-average to weight-average inolecular weight of about 1.8.
z. Studies of the physical and chemical properties of heparin degraded by' acid hydrolysis so that about half the anticoagulant activity is removed, lead to the con- clusion that in the initial phases of this hydrolysis the formation of internal esters l n a y occur .
3. Similar studies performed on a heparin sample submitted to periodate oxidation to a degree of degradation similar to that of tile hydrolysis degradation, reveal that more drastic changes occur in this process. These changes involve: the loss of N; oxidative cleavage; and the reduction of molecular weight.
INTRODUCTION
Molecular weights of heparin of from 7600 to 20000 have been reported from measuremeuts by diffusion 1,2, light scattering3,L ultracentrifugation a,~-lz, and re- ducing group assay1:U 4. Correlations between ttle molecular weight of a heparin sample and its anticoagulant activity have been claimedS, ~ and denied a. This discrepancy seems to be ttle result of either comparing the activities of a series of fractions derived from a sample bv fractionation 9 (correlation confirmed), or by comparing the activ- ities of a number of different commercial heparin samples a (correlation not found) with their molecular weights. Even in the former easel however, the activities of the fractions varied not only with molecular weight but also with sulfur content.
It is therefore felt that one must study a heparin which consists of highly uni- form chemical and physical properties. In order to do this one must not only de- ve.lop effectiw; fractionation procedures, but nmst also be able to measure the de- gree of heterogeneity. I t was the purpose of this research to investigate and de- velop a number of physical and chemical methods for characterizing heparin samples. Since a previous a t tempt at detecting heterogeneity by e.quilibrium sedime.n- tation had not met with success la, this work concentrated on the use of equilibrium
" This is contribution No. r57 of the Inst i tute of Cellular Biology, University of Connecticut.
Biochim. tliophys. Acla, i58 (I968) t o3 - r I6
1o 4 v;. BI{.kS\v:-:;.
sed imenta t ion f()r the measurtqF.,ent (~i ~,~olecular weight and bcterv)genei!y.."~s :. result, it ?~as bee:~, hmp.d tha t >revi(~usiv publ ished moltoeft, : wt'.igi:ts :~.i i,.'t~a'.J::' de te rmined by uhracemri[ugatio;~, arv~ incorrect, and are to,~ low. . \ ! t l '~ )vgh t!:i: investigation: d()cs not set t le the mo!e~:u!ar weight- .ac t iv i ty .~'orrelati~m q~.wsti~>~:, :' d . ~oes more carefully scrutinize the meth )ds i~.vo!ved in determining ,~w.ie'.'v.!ar wt i!,41
by centr ifugal methods, tn addi t ion, some a t t en t ion is paid t,> the p~cchanisp,: i)\ whici: hxdr(~lvtic and oxidaLive degrada t ioa takes place.
M E ' t ' I I O 1 ) 9
H@arin co~zcenlration Hepar in is ex t remely hydroscopic, aud wi!l absorb moisture up to 2o % of it~
d r y w e i g h t . T h e amoun t absorbed varies both with tim ] tumidi ty and the physicM iorm in which the hepar in is prepared . As will be ..et.n, . s ,, i t is diflScult to reln()ve at] th~ m~Hs- ture wi thout degrading the. mater ia l . Fur ther , because one usual ly works with hcparin solutkms conta in ing I M NaC1, dry weights are not a feasible, means for de termi~ing hepar in concentra t ion. Because most labora tor ies have Kje ldah! equipment , the de- t e rmina t ion of ni t rogen was favored for de te rmining the q u a n t i t y of b, eparin, e.x~c~. though there is more S than X present hi heparin. However, sin"e the quan t i t y ,;f N
.. . " ) r . present per mg of dry heparin varie~ front .~amDie te sample, it must be. known ~ e:~)~ e one can de te rmine the coneer: t rat ion ~[ hepar in in solution, l -xper iments were p(w- formed in which a hepar in sample was dried at I~o ~ ove.r P.,O.~ in a vacuum d r \ i n < pistol. A por t ion of th is sample was removed each day for 7 days, weighe.d quickly, and the N eonte~:t d{ retrained. The quan t i t y of N present per mg t,f "dry" heot~rin was found to rise as the moisture was removed, reaching a m a x i m u m on the m-v.i vv7 y :d dav. Thereaf ter a gradual, decli,ue in N content comlnenced, r eacb i rg a level of ab~.,ut 90 o.,o of. maxxmunt- ' by.. the 7th ,.lay. Ant iccagu!an t assay show<:d a de.crease in acti\ ' : ,tv of 2 o % by the 7th day whi]e reducing group assay showed an increase, c~f 35 %: thus in(licating, tha t considerable degrada t ion occurs under *;~,~ ,.'onditi has "l'!~e same t y p e (:f cxper imer~ wa.,.' ~:erf~rmed at 8o ' , whereupon the X to dry ' " ~ rat io was again found to increvse, reaching a maxi:m,.m on the 3rd day. This ti~:;e
? + t , ( - however, the auc<'eeding declir;e was bare ly discernible by the 7th day . ] a r ~,,i, an t icoagulan t assay indica ted no decrease in a<:tivitv by "ci~e 3rd day. Therc[<,rc, ~h(: technique o[ de te rmining the q u a n t i t y of N t,'resent in a sample of hei)arin \vbh:h was dr ied at 80 ° for 3 days m~.der \.'3.CUUlll over P,,O~ was adopted . Om:e the N c~ntc,:t for a d ry sap, tpic is klloWi1 for ~t g yen hepar in , one merely determines X c<) ~.t,.:~t ,~f an unknown solution of this heparin and can then calcula te the hepar iu concentrat i~u Most sanaples were %und to contain about (i.0220 - • 0.0001- Ill S N:':ng hcparin.
Refractive increment For l ight sca t ter ing measuremenis it is necessary to know the refract ive in-
c rement of hepar in in the ~ M NaCI solvent in which it i~ s tudied. Shlce it is not de- s i rable to d ia lyze solutions (ff hepar in for fear oi loshlg a port ion o[ the ].ower m~le(.:ular species, the refract ive increments :necsured by means of a Phoenix Pre.cision D i f -
f e r e n t i a l Refrac tomete r mus t be corrected for diluticm due to the moisture in t roduced by the sample. Fur ther , it was di~cov(~red tl~at tiw. usua[ pract ice ( / ob ta in ing a refract ive index ditterence between a z % solution of po lymer and ~Avent, and ti?en
t?iochiw, t3iophys, Acta, 158 (1908) io 3 -T 16
HEPAI,:IN: MOLI';CUI.AR WEIGIlT AND I)I,;GRAI)ATION 10 5
dMding it by the concentration of the solute (a simplification of proper procedure), was somewhat in error. This became evident when a graph was constructed of re- fractive index difference vs. concentration for heparin solutions in I .~'[ NaC1 over a concentration range from about o.15 % to 2 %. Such graphs were always linear. Frequently, however, the line did not intersect the origin, but missed it by about i -3 % of the refractive increment. The refractive increment was taken as the slope of the best line through the data at the concentration of interest.
Specific volume Specific volumes were determined by means of a density gradient column
containing a gradient generated by a mixture of heptane and bromobenzenO s. Great care had to be taken to saturate the column with a NaC1--H~O solution of approxi- mately the same total chemical activity as the aqueous heparin--r M NaCl solution being studied, in order to prevent hydration or dehydration of the unknown sample droplet as it descended to its isodensitv level. L.'sing standard solutions of NaCI, the densities of which were known, it was possible to bracket the unknown droplet with several known droplets and to interpolate the density of the unknown by means of a cathometer. Calculation of the volume increment contributed by the heparin in- cluded a correction for the dilution of the solvent NaC1 by the moisture in the heparin.
In analogy with the determination of the refraction increment, a graph was constructed of the volume increment contributed to the solution by the dry heparin vs. the concentration of heparin. The slope of the line through the data at the concen- tration of interest was taken as the specific w)hune. Such graphs were linear, but in contrast with the refractive increment studies, usually intersected near the origin, indicating that the specific volume of heparin in x M NaC1 is independent of concen- tration over the concentration range of o.15 to 2 %.
A nalyses
The anticoagulant assays were performed by South Mountain Laboratories of Maplewood, N..]., in accordance with the USP XVI1 in vitro method. Elemental anal- yses were carried out by Weilcr and Strauss Microanalytical Laboratory, Oxford, England.
The number of reducing groups in heparin were determined by the production of Prussian blue in a ferricyanide reduction methodlL The equiwtlent weight~ of the various heparin samples were determined bv a methylene blue binding method similar to those of COPI.EY AND WII ITNEY TM a n d MACIN'I'OSH 19.
ULTRACE N TI2IFUGI,; /'2X PE i~: I.M E N'I'S
There were 3 types of ultracentrifuge experiments performed ; they were velocity sedimentation, equilibrium sedimentation, and the Archibald method. They were ail performed on aqueous solutions of heparin (o.i5-i %,) in I M NaC1 at 2o ~' in a Spinco Analytical Ultracentrifuge using Schlieren optics. None of the solutions were dialyzed for fear of losing low molecular we.ight components through the bag.
The velocity experiments were perfl)rmed in the usual manner at 5978o rev. per rain. The sedimentation constants reported are. those found in r M NaCI and were not corrected to standard conditions by the SVEI)BEI{G ANI) PEI)FRSON 20 method,
Biochim. ldiophys. Acta, T58 (t968) ro 3- I)6
! 0() i:1 )~R.kFW}.:.~.I
because the specilic volumes <)f N:e !!cparins in salt-free water \vt.::e xmc krow:: Sed imenta t ion equi l ibr ium run : were performed rout ine ly at 9945 re\'..hv, h~. ~%nd
were found to have a t ta ine , I tnac t ica l equi l ibr ium within 48 h. tqowever, ~iI rtv.~s were carrie.d out for 7 2 h. 3 ° l?llll d ~ ) u b l c sector cells we.re used, tilled t~ ab.out :~,:ii o their capac i ty with solution and reference s o l v e n t . . \ silic(me oii \v~-s used to establish an ar t i l ic ial bo t tom for the cell. Ryes per formed at 15 220 re.v.,.min were found t(; gi ec in 24 h (,f centr i fuging the. same weight-average molecular weights as those at 9945 rcv./mirt. However , measurements on the p la tes were more di!Ytc'dh t(~ make, and Z-average nlolecuiar weights were fmmd to be somewhat less than th~se obta ined at the lower speed. I t was therefore [elt t ha t 9945 rev. /min was tiw. best sf)ccd f<~, this work.
The image on the plate was in'ojectc d b y an enlarger onto graph paper s~) that the total hor izonta l P..lagnitScation due t(.' u l t racent r i fuge optics ami the. enlarger was ten-fold. The image was t raced o~. the g raph paper and readings o[ the X and V coordinates of the Sehliercn curve we.re made amt put on punch cards. The: apl.)arc'nt molecular weights were cmnputed with an IBM 7o4 o computer , using a F~wtran If p rogram developed in this l a b o r a t o r \ . One of the equat ions zt used in this pr(~gram was:
where .dc is the concent ra t ion difference across the cell which is ob ta ined throug".. knowledge of the area under the. Schlicren curve, the refractiw'~ increment, ~md the opt ica l cons tan ts of the ul t racentr i fuge. % is the init ial coucentra t ion, and :v,,~ and Xb are the d is tances from the r(~tor to the meniscus and bo t t om of the cell respect ively. All o ther symbols have their usual meaning. As VAx H(~I.I?E ez showed, this eqmtt ion yields an appa ren t moleculaT weight at ti,.e eoncent ra t iou (oh l_ c.,~)/2 (\vldch is a lmost equal to c 0 under these exper i lnen ta l conditi~ms).
Another equat ion ~" u s e d . \V~ts:
=[ l fdl.q 3.lw 6,,s(~ 7:,-p;j L ~i-x'-' J , I[~
where c is the concent ra t ion at the d is tance x froln the rotor. ".I'he weight average molecular weigi~t a t any point in the celt is ca lcula ted as the slope, ;,~ the point, cf a graph of ]nc zs. x 2. In pract ice the sl,)pes were almost linear, a:~d the slope of t l 'e bes t s t ra igh t line th rough the d a t a was used. in order to usa this equat ion one must know the concent ra t ion at some point in the cell. This can be got ten by ei ther o':j 2 equat ions :
el'D_ - / l C . / ( ( . ~'IC/CO - - - I 'I { I i 1 :
(see ref. 22) or
%Yb [ X b - j ' ' " -- I ),'- .IC]"(
c~, ~.0 . . . . x = - j - - (t\'.,
(see refs. 23, 24) Eqns. l i t or IV give essent ia l ly s imilar results, and when used with IT yield
molecular weights in good agreement wi th those ob ta ined from I. The combinat ion
Uiochim. 13iophys. :~cta. t 58 (1968) :to?, t ; 6
HEPARIN" MOLE('ULAI{ WEIGIFF AND I)EGRAI)ATION lO 7
of IV and I I gave practically identical results with Eqn. I, so the results reported are those which resulted from the use of Eqn. I.
The Z-average molecular weight was determined using Method 2 of VaN HOLDIC aN t) BALDWIN e2, which involves connecting tile end points of a graph of (I/X) (dc/dx) vs. c. The equation of this line is:
• / J 7 -- - - L( .o2(1 - - 9 p ) J ( V )
which yields an apparent Z-average molecular weight at the concentraton Cm ~-cb (which is approximately equal to 2 Co). VAX HOLDE AND BAr.J~WlN 2~ state that if such a graph is linear, implying small concentration dependence, the slope of the best line through tile data can be taken as equivalent to connecting the end points. Since our graphs were ahnost linear, the Z-average molecular weight was obtained from the slope of the line.
The program also enabled one to plot the slope of Eqn. I I at any point vs. x, and to plot I/XC dc/dx vs. x, both of which are inore sensitive to molecular heterogeneity than any of those mentioned above. A graph made by either of the latter 2 procedures for an ideal homogeneous polymer yields a horizontal line tile height of which is proportional to the weight-average molecular weight at any point x in the cell. These are far more sensitive to heterogeneity than a simple graph of Eqn. Ii .
Once the apparent weight-average molecular weights were known at the various initial concentrations, another program was used to calculate the true molecular weight using the equation of \ViT.l.ta.~,ts et al. "-~ based on a study by Jo~Ixsox, Kr~aus Axt~ SCATCH:~m.) "6, for the sedimentation of a polyelectrolyte in a solvent containing a uni-univalent strong electrolyte. The equation is:
[i EMs(I--~TsO)] [, EMs(dn/dc)s]
Ma = My" . . . . . . . . . . . . . . . . . . . (VI)
1 2 -'~PL'8 J t
where E is the charge on the polyelectrolyte nlolecule, MA, MI>, and Ms are the ap- parent molecular weight of the polymer, the true molecular weight of the polymer, and the molecular weight of the salt, respectively. The other terms have the usual significance for either salt (S) or polymer (P). It can be seen that a linear graph of Eqn. VI may be constructed in which l iMA is plotted against cp. The slope of this graph divided by the intercept has a value equal to:
S l o p e E'~Ms . . . . . . . Sx . . . . . . ( V I I ) I n t e r c e p t 2 M pcs
For a I M NaC1 solution this becomes:
5./?'2 Mp =. . . . . iX ' t i t )
SI
The computation proceeds by assuming a value of E, finding M r from Eqn. VIII , then using Eqn. VI to calculate a value of M a (and E), and finally comparing it to the graphically determined MA. By reiteration, the procedure is continued until the
13iochim. [3iophys. Acta, r58 (1968) IO3-116
IO~ i{. I-~}{.\S\VV L~
2 v~dues of .]IA are identical. The lasv a~>umcd valu'e ,~)~ ] ' , and the resuhin~ vahm :;! Mt, are tile c~>rrect values f~)r those parameters.
For the Arc]ribald nmthod <)[ -btcrnfining tile molccuta.- weight< 3o *::ST': double sector {'ells filled with solvent and solution were ccntrihlged %r z h a~ eat!. o ~, 23 speeds; r 5 22(; roy./rain, 3 ~ 44o rev.:min, and 5o74o rev.:'min, ,.vitho'at stopping the centrihlge be'~ween speeds. !.'i.':t':-re~. were taken throughout thia time. Ti'.=is procedure permit ted the s tudy of (dn/clx)m from on: --= co to c m = o. Alter trac 'ng the el"dargec] image a modified "Fm~vv.~mx a: p l , t was made, in which (dn.'¢ixbn, ncrmg,.~ized f(,~ speed and schlieren ,~.ngle, was plotted against the area of tl~e peak, each pict,,i:-<: producing a single point on the graph. The best line "d,,nmgh these t;oin:s yields (dn/dx)m h~r cm--= co at the Y intercept, and tile: totai alTea c,[ the boundary (pro- portional to q~) at tile Y intercept ((dn,"dx!n, --- oi. The slope cf this line at lhe Y intercept is proportional to the al)parent weight-average n~obcuh: weight of the heparin at %. It the line is straight this slope nmv be obtained by simply dividing tile Y intercept by the X intercept. The resulting :tpt)arent m,~[ecuia.: weights must also be corrected for charge b \ t lm methc;d outlined above using Eqns. V i--\ . i iI .
L i g h t scal ter in~ m e a s u r e m e n t s
These were performed by mean~ of a 13rice-.Phoenix ligt.',t scattering phot, , :nete, on solutions of heparin (concentration range O.15--2° 'o i ill I M Na(.'i <;;" I ~1" Ya.[. The solvent and solutions were clarified by filtration through a series of Millilmre filters ending with a V F W P fi l ter . . \ check on the light scattering t~:chni¢iue was made by determining the molecular weight of bovine pancreatic ril~o:.mclea~e :\ (\Vorthing- ton RAS). It was found to be 13. 7 • Io a,
Viscos i t 3, sgud.ies These were performed in a Cannon-Ubbelhode Dilution viscometer (size 5o)
with a solvent time for z .~] NaC1 of about 26o .~ec. [t required ;:. minimmv of ab,~ut 7 ml ()f sample :-rod had a dilution range of about 8 to r. tn practice, 8 ml .'~[ sample and a 6 to I dilution range were utilized.
l)egradatioJ~s The heparin sample that wa~. to be degraded was first :tia':vzed h)r several dav.a
against many changes of distilled H._,O. I t was then freeze.-dried, and an approx. ~ % solution t)f this heparin was made up in distilled !l~O. The pt l of a portion of thi~ solution was adjusted to 2.5 with acetic acid (about z I °o), and it was kept at 7 °.: for 12 h. The product was then co~,led, the acid neutralized with XaOt{, and it was dialyzed against several changes of distilled 1-12(.) r,,r several days and was flnalh" heeze-dried. The anticoagulant acth, i ty was fmmd to be abmtt 6o % of the original; undegrade.d heparin. Another portion of the original ~ 'Yo heparin solution was oxidized by per%date at room temperature fi)r 3 o h in the dark. The pr¢~duct was recovered in a mannc.r similar to that (~f the hydrolyzed sample, and was found to have retaiued about 45 % of its ant icoagulant activity.
RF.SUI.TS
P h y s i c a l s ludies
M o l e c u l a r weight measureme~zts. As can be seen in Table [, the agreement between the molecu!ar weights obtained from light scattering and those obtained from equL
]3iochim. t~iophys.."I eta, 758 ( t 968) I o3- ~ r 6
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ti[~rium sedhlmntati~m is satisfaclorv c(msidering the ex~)erimen~ca} difficulties in- volved. The results, however, of the Archibald method were in s~.ich poor agreement with those obtained from light scattering and equilibrium se(limentation that they were considered almost worthless, and therefc)re are ne~t reportc.d. Yp~i.\.~r~ 2s l)oinks out tha t tile TRAITT?,IAN method leads to a mole :u!ar weight ~tvp, l a g e t i e : a c t tr) :.t Z-average than a weight-average. Comparison of ~u: results, however, with the Z- average values obtained from sedimentation also was l)oor. The etr()rs seen:ed t< arise from the difficulty involved in estimating the height of the schlierc:~ line at the meniscus 2", and then again later in the extrapolat ion of the modified T~.¢:xl:rst.~x plot to c =- %. This metht)d .might be usable f(:r electrolytes of i(:w molecular weight with some prae.tice, but tlw equilibrium method seems Jnhcre:~tlv n:orc at'curs>:, and hence preferable when the duration of the experiment i~ ,.l¢~r a consideration. Of interest is the fact that the magni tude of the correction of the apparent weight- average molecular weight to) the true. weight-average ntolecuiar weight is or~ the average about I . I6. It attaip.s, ht~wever, a value ~f r.54 in the case of the het~arip. dissolved in I .M Nal. Fair agreement was found between the 2 weight-average molec- ular weights obtained by equilibrium sedimentation of the Riker hcparin when d}s- solved in ~ M NaI or ~ M NaC1. Tb.e fact that the apparent nmlecu!ar weights of R!ker hc.parin in their 2 solvents are quite ditierent is an indicatJ~m t)f the impt)rta:lct'. ()t the \Vn.tI:\ .~s ,,'t aI. 2:' correction. The Z-average molecular weig~ts are felt to be much less reliable (perhaps d-2o %1, as the apparent Z-average molec'fla; weights a-suailv appeared quite scattered when plotted against concentraticm. "i'ho siune coi~.sider- aliens would apply to the weight ave.rage and Z-average :ran'bet of charges..[z~ general, however, the Z-average molecular weights el the commercial and degraded heparins are a little less than twice the. weight-average molecular weigb.ts. It was observed that the degree of heterogeneity observed from plots of tim slope (~f Eql,,. i [ agreed quali tat ively with the Z- t() weight-average molecular weight rakio.
There seems to be no relation i)etween the me!ocular w('igb.t ()r the number (~f charges an.'t tlm anticoagulant activilv of the hepari:,!. Further, there is z~.othing in the physical da ta to suggest a c!ear difference between :nticous anti lup, g heparins. One can see, hom tlm table, however, th;tt partial hvdrol\ 'sis caused no loss of mole(> ular weight within ti~e limits of erwr, as opl)osed to parti-d ,~xidation.
Other pkysical studies. As can be seen from "['able I, there is no obvit)us relation between anticoagulant activi ty and the. refractive increment, tl 'e specific volume,, or the sedimentation coefficient. In the ' - (i%raded series, how,'~\.er, :.t is interesting i~" note tha t the partially hvdrolvzed sample has a se.dhnentatio:~ cc~eff,.ciel~t less than the original parent material, while tile partially oxidized sample tms a t:igb, er one.
The intrinsic viscosities of the. various hcparin samples were studied in 2 dif- ferent solvents: I M XaC1 and o.o 3 M NaC1. The purpose ~)f this was to at tain son> measurement of the degree el extendabil i tv o{ tile molecules by decreasing tllc ioni(: strength and thereby allowing the repulsive forces to act on * he heparin c'.'.~aip. Thus, the ratio of the intrinsic viscosities at the. lower ionic strength It) tha t at the higher, represents the degree to whict} the. molecule can expand. This in turn will de~)end (m the flexibility of tile molecule and the number el charges (m it. Except for an intm- esting decrease occurring in this ratio ~zpon hydroly.sis, the ,.esults 4o not allow any generalizations about heparin type .:)r relation to activity.
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Chemical shedies The results of the cb, emic'd st,ldics are summarized m "Y~,.blc II . "The imi)ort;m~
observations will be pointed ,out helTe., bill their signi{icance will bc covered i~: l;~s- C I - S S I O N .
The reducing ability of tl,e. c~)!'m:ercial heparins studied here varied bctw~,~,~l o.87 and 4.7:r mg of glucose..'g (;f heparin, and the .methylene bin,,: bindiag e(tuivaiez~{ weights were between i63 and 186. "file.re seems t~ l;e n~) correlati(m between a,.~ti- coagulant activi ty and either of these parameters. Xeitl.er d(, the r.,~easurement> distinguish between nmcous and hmg hepa.rins. I t might be p, oted, hm~wever, that th'c oxidized sample is distinct!\- different in its redudng sugar titre, while the hvdro!vzed sample has a higher ectuivalent weight than its source.
"l'he elemental analyses viehl few obvious differences bct\vce:l t-eparin samples. However, it is interesting t() not(: tha t except ill one case, the N c,,mtent of all th(, hep~rins was less than that predicted fr()m the prop()sed lraparin structure a~;. All <'f the C and I[ contents are higher and the remainder contents (presumed to bc Na and (.)) are h:wer than the theo r \ would iv, ad one 1o expect. Of the d'egraded hct.,arin series tile oxidized samples have lower N c(mtcrlts and the: partially oxidized sample has a distinctly higher S and (2 content, with lower If and remaipder c~mtents thin? either tile source hcparin fr~)m which it came, or the: partially hy.qrolyzed aam!;ie
D I S C U S S I O N
Physical measureme~zgs All 3 of the principal techniques were used in past sedhnentation studies,
i.e., velocity sedimentation-difiusiona,~u, '°, u, e(luilibriuml"-',ia~ and the Archibald method s,9. While all the. investigators used swamping elcctreiytc, only f,.\sIg::3d:' in his Ph.l). dissertation reported the use o f a correc.ti(m factor lot ti~e sedimentation of polyelectroiyte. In the subsequently published paper ~, however, mention of riffs correction is omitted. The neglect of such a correction in equilibrium or Archibald studies of materials of such high charge "s serious. Assmning the polyele.ctro!yte has a molecular weight of uoooo, a charge of zoo, and is dissolve.d in r M NaC1, the co,e- rection fl)r the api)arent molecular weigl:.t couhl be as large as 47 %. That i~, the extr;~- polated apparent molecular weight would have a \ a iue of only z3(.'oo. Undout:)tcdl), a similar situation holds with velocitvsedimentation-diffusion studies, but this problem is somewhat more complex (see for example refs. 20, 3~, 32).
The application of ]dqn. VI must be handled with care. "Yhere are 2 terms m the numerator of the equation, one involving specific volumes and th,.: other i rvo!ving refractive, increments. The. first term arises because tile polyelectn)lyte and the s?.h distribute themselves differently in the cell during scdimentatiou, generating a ~
internal electrostatic field. The second term is due to the Donnan equilibrium, and is a result of lhe more concentrated solutions of polyelectrolyte "pumping" electrolyte toward a region of the cell v:here the polyclectrolyte concentrat ion is lower. Optical systems of detection measure the refractive index, ()r its gradient, of both solutes ad(litively, and do not distinguish between polyeleetrolyte, and electrolyte. Sub- traction of the solvent gradient by using a double sector cell do~es not correct fc~r this Donnan effect, because the salt is distribute.d differently in thc reference (:ell without the polyelectrolyte. This subtraction should be performed, however, since E:tn. \ ' i
t~iochim. 13iophys. Acla, r58 (19~.)8) zo3 • r',i
HEPARIN : MOLECULAII WEIGHT AND I)EGRAI)ATION 113
was derived assuming that a salt-solvent base line would be subtracted. If a poly- electrolyte is dissolved in electrolyte, then dialyzed against the solvent, and a measure- ment of the refractive increment is made of the dialyzed polyelectrolyte vs. its dialyzed solvent, a low value of d1,/dc will result. This arises froln tile fact that the Donnan equilibrium established during dialysis has "pumped" salt out of the bag. Therefore, assuming equal volumes inside and outside the bag, one is measuring a difference in refractive index between a salt solution of concentration cs : Acs, and a polymer--salt solution of total concentration Cs--Acs -t- cv, where cs is the original salt concen- tration, and Acs is the increment "pumped". i t can be shown that this low refractive increment, (dn'dc)a, is related to the second term in Eqn. VI in the following manner.
(d!~/(ic),l .... [I EMs(d~/dc)s]
Therefore, if one performs a synthetic boundary run using the dialyzed solvent and solution, and then uses the area of it to represent c o in a calculation of molecular weight by a method such as that in eqn. I, one has automatically corrected the data for the second term in Eqn. VI. LAsKJ.:~ ~s (and presumably I.:\SKER AXD S'rIVAI.M ~) did this, and then applied Eqn. VI, thereby correcting for the Donnan ternl twice. In summary we would state that all ultracentrifuge studies perforlned on heparin so far, except that of LASKEI,I 1'5 and L..\SKER AND ST!VALA 12, are low, whereas the latter two studies are probably a little high. Although there are difficulties involved with light scat- tering also, it could be expected that such studies would give a more correct result than uncorrected ultracentrifuge studies. In this respect it is interesting to note that tile light scattering results reported in the work of BAm.ow, SANDERSON AND McNEn.l. :~ are x.o 9 and r.z 4 times the respective values determined by sedimentation-diffusion. The average correction found in our study for equilibrium runs was z.I6. These cor- rections are much lower than the known charge on heparin might indicate, suggesting that not the entire charge is effectiw~.. This may be due to lack of dissociation or some similar phenomenon. In this respect it might be noted that the equivalent weight of heparin determined by electrical conductivity :~3 was found to be 665 instead of about z7o-i9o as determined by titration or dye-binding methodsg, :" (see also "['able [I').
Tile polydispersity of heparin is considerable, since the average ratio of the Z-average molecular weight to the weight-average molecular weight is about 1.8. Since this ratio should be only about I. 5 for the "most probable" distribution, i.e., that which results from a linear condensation polymerization or tile random cleavage of a uniforin, highly polymerized molecule, one might conclude that there are several unresolvable but distinctly different species present. Our detection of heterogeneity by means of equilibrium sedimentation, in contrast to I.ASV.ER la, is probably due to our use of h)nger solution colmnns, lower speeds and a more sensitive graphing procedure.
I t must be pointed out that Eqn. VI was originally derived assuming an ideal, monodisperse, strong polyelectrolyte. Since these conditions are not fulfilled bv he- parin the weight-average molecular weights reported are t)robably not correct. They are, however, more correct than those reported which did not use such a correction for the polyelectrolyte nature of heparin and t)robably are useful for coml)arative pur- poses. The fair agreement with the light scattering data strengthens this conviction. The application of Eqn. VI to the calculation of M~., however, is of greater doubt and
.Biochim. Biophys. Acta, I58 (z968) 1o 3--II6
I 14 !c i~R:x~wiCl.i,
the resulting values can be considered c~nlv crude estimates. "['h<'re m;-v be s(;:nc ~l:~,~ in them, however, %r the calcu!ati(H" of .l/z..'3/w %r comparing ¢~::t, heparin sa:'.'q;ic with tha t of anoth~r in order t(~ estimat~ tht~ir relative hetcroge:,?eity.
Chemical mea.sure]nents A certain portior, o[ the variatio~ c){ the elemental c~)nt~mt t~robably (:a~ be
accounted for by variation in the mmfl)er of acid gro'.aps neutralized. H.c, wever, tI;e heparins studied here contai~ higher S.'N arm ( ' :N =atios ti:an eith( r ~)f the 2 propo~:d formulas}~, u°. The equivalent weights and the C.'S ratio have va!m~s about :nidwav between those that might be expected from the 2 str~.ct',m~s. {f ~m material is c(m- tamir~ated with an S-containing i)olysaccharide lacking in X, with a higiwr equiv;deat weight than heparin, then the structure, pr()posed i ) v ]~RIM:kt.;t)MI'IE AND \:VEi~,B}%I{ :'~'), or one with an even higher degree c;f s~fifation, would be accept;:lfle.
Degradation ,~tudie.~ .]ENSI.'N, ~.NI,'LI.MAN AND ~",'L\'i'IN 6 ,,)bse.rved that mild acid !w-trolvsis affect~
the molecular weight of heparin very sligbt!y while decreasing the frictional ratio <;f the molecule.
I.ASKI:.R ANI.) STI.\:AI.A '2 recenth" found that samples which had up to 75 % of of the anticoagulant acti\.itv removed by mild a.cid hydr,)lysis showed no significant differences in the.ir molecular weights, intrinsic viscositie% (~r other physical, properties when compared with the :mdegraded mater ia l i:urther, there was no detectable change in the N ~md S contents.
Tables I ai~d I I show that acid h\'droh,sis which destroys approximate!y l~alf the anticoagulant activi ty causes little change in ti~e molecular weight, intrinsic viscosity (in ~ M NaC1), or elemental analyse~. Notable is ti~e increase in the equivalent
1 ) , . weight, h()wever, rcprc, e_ltm~a, " ,, a ,< ~ of about r3 charges from tim molecule. If this charge loss is due to sulfate hydrolysis, this wouM result in a decrease of m~)ie'.:ulaT weight of about K~2o, ;t figure width, takillg all the uncertain;ties ~f the rr:~flecnlar weight determination int(: account , may be :tetectable, but was not {fl;served. : \ t least one can say tha t little or no ~:hain scission occurs. These ¢;bservations ,nay als(~ answer the question ~)f whether the released S is present as bound H..aS() ~, since the bound a(:id could n()t contribute to the ;n<~!e(:ular weight mea~m'emer:t.~ though (.bring so in 5 analx'scs. 5o one might c<.;nclude that acidity is de.str(;ved without rem(~ving S, N, or C ~tt()ll-s. t h i s could c()m(', about through tt~e formati~m of internal esteFs. "I'hi~ would then explain: (I} the unchanged intrinsic visc~sitv measure,'t in ~ .~L Na('i, and possibly the decrease in frictional coefficient observed by .JI.CNSEN, ~NELLM:KN .\N-~ SYLXT.Na; and (2? ~hc ~act that the ratio of the value of the intrii:sic viscosity per- formed in o.o3 M NaCI to tha t per((nmed in t M NaC1, is close to uni ty being consider.- ably reduced from the value of r.,t3 observed for the uni~ydr()lyzed (;r the t.33 observed for the oxidized. Undoubtedly the reduction in charge contribute~ to this effect, l~.~t the restrictive nature of the. bonds that art' formed may have the larger effect on this ratio.
Opposed to this is the work of H~.;LB~.R'." ;\X;) MAmx~ :~ who found by titration techniques that the auto-t 'vdrolvsis of heparinic acid removed X-sulfate gronps twice as fast as (_)-sulfate groups. While they took excellent precautions to prevent h;mding of released acid to the heparm, the\ ' unfor tunately (lid n o t p~:r{orm elemental analyses
Biochim. Z~ioflhys. Acta, I5~ (rq~i,'4) ~o3-~1(~
HEPANIN : MOLECULAR WI';IGI-1T AND 1)EGRADATION I 15
or anticoagulant assays throughout tile course of degradation. Therefore it is im- possible to determine whether the various studies, i.e., LASKV:R AXD S'rlVaI.a 12 and HzI.B~lcr :xx1~ Ma1~IXI n and the present investigation, took place at comparable stages of degradation.
As (:an be seen from the tables, the proi)erties of heparin are drastically affected by a periodate oxidation which had been carried out until a little over half the anti- coagulant activity had been destroyed. The most obvious change involves the in- crease in reducing groups from approx. I mole of reducing sugar for every 77o0o g of heparin to i for every x4ooo g. Since 2 reducing groups should be produced by each oxidation, this indicates that on the average about every second l mparin molec- ule has been attacked. Since the activity has been reduced about half, this would seem to imply that such an attack destroys all the anticoagulant activity of that n~olecule.
Caution must be observed, however, for oxidative cleavage of vicinal hydroxyls is not the only process occurring. The N content is lowered (probably by oxidative cleavage after N-desulfation), the S content is raised (by the preferential elimination of other atoms), and the molecular weight and the number of charges as measured by sedimentation and dye-binding are decreased. This indicates tllat some chain breakage has occurred ak)ng with other reactions. From Table II one can see that some II and remainder atoms (O and Na) are lost. These losses make possible the higher S content observed. Actually, a large part of the decrease in molecular weight can be accounted for by taking into consideration the number of charges and the amount of N and remainder atoms removed. A very crude calculation which assumes, pre- cariously, that all the ()-sulfate groups are left intact by this process, leads to the conclusion that a inolecule of molecular weight II3OO , when stripped of the above atoms in their indicated proportions, attains the chemical features and molecular weight (about 84oo) of the oxidized sample. The fraction I 13oo/I 25o0 would represent the fraction of molecules cleaved. One must look at such a calculation with appre- hension for several reasons: (I) one is using the weight-average molecular weight as if all the molecules were of that same size; (2) some parts of the calculation, such as dye-binding equivalent weight, involve number averages ; and (3) O-sulfate is probably hydrolyzed to some extent. The conclusion, however, is probably valid in a quali- tative way. That is, although a sizable fraction of the molecules have suffered oxidative cleavage at a pyranose ring, most of the polymer chains have not broken, chain in- tegrity being maintained through the pyranose oxygen. The not so great decrease in the intrinsic viscosity and the extendability, or the ratio of intrinsic viscosities at o.o3 M to that at I M NaC1, seems to confirm this picture.
l 'inally, it nmst be remembered that these preliminary studies were not per- formed on salnples which contained highly uniform molecules, and that the conclusions arrived at must be considered tentative. Undoubtedly, such degradive studies will have to be repeated when sharp fractions of highly defined molecular and chemical properties become available.
A(;KNOWLEI)(;EME NTS
Tile author is indebted to: Prof. M. STA(;EY, l ?. R. S., whose hospitality made possible the initiation of this work at the Department of Chemistry, University of
Biochim. Biophys. Acta, 158 (1968) I o 3 - I ~ 6
I.~0 }. BRASV~'T LI,
l ¢ i r m i n g h a m : D r . A. B. ',:os;'~-;i<, for h e l p f u l a d v i c e a n d m a t e r i a t s ; Mrs . LOR>.:\~X::.
}'~()'i'H, ~_1.I1([ ;& nUIIIB('F of o t h e r s w t : o aI)l}" ~_lqsiSic(l in t i l e m e a s u t c m e ~ t s ; a n d ~d>c)\<
all t o l ) r . ( i . A . Crll,l~ER'r in w h o s e l a b o r a t ( ~ r v t i f fs w o r k w a s b e g u n av,,t whc)se s u g -
g e s t i o n s c o n t r i b u t e d t o tl~e b e s t in t h i s p a p e r .
T h i s i n v e s t i g a t i o n w a s s u p p o r t e d in p a r t b \ : P u b l i c He, a h h S e r v i c e Rcsea : c}"
G r a n t s N o . 8 2 5 8 a n d No . 8 8 9 7 , f r o m t h e N a t i o n a l H e a r t I n s t i t u t e .
T h e c o m p u t a t i o n a l pa ' : t ~f t h i s w o r k w a s c a r r i e d o u t in t h e £1"m~puter C e n t e r
of t h e U n i v e r s i t y of C o n n e c t i c u t , w h i c h is s u p p o r t e d in p a r t b y g r a n t ( iP -zS~! ) ~t
t h e N a t i o n a l S c i e n c e F o u n d a t i o n .
R V,L.'E I(1: N CI.S
I ?d. L. Wor, t,',~oM, l(. 3h)x'r(;oY*LRY, ] . V. I'L.\R..XmXOS AXD L'. l~.,\rn<;~m, J . A~n. Chem. Sot., 7 z (x95 o) 5790.
2 K. II. ',MF,¥Ya, .\:atural and ,5y~elhetic High t'olymc;,s, in terscience, New York, 2p, d ed., i95 o, p. 45{;.
3 G. 1-1. r3Am.ow, N. D. S~xxut~asox .xxt) P. D. McN£II'.L, Arch. Biochem. t:;iophys., 94 (I9()U 5 I8 4 F. ASCOI.I, ('. l-~o't'Rl5 .rX Y t) ~k o ~2 . LIOU(.)RI, J. ]Htys. Chem., 05 ( |961) [99r. 5 A. (;R0XV,'.\LL, B. IN<;LEMAN AND II. ?,lOSl.~l.~.XX, Upsala ].(ikaref6r~:~z. Fdrh., 5o (19451 397. 0 F,. jv:t~sg, g, O. SXF, I.L.~LaX AXD 13. SVLX.'t~X, ./. Biol. Chem., I74 U9.t8/ 2¢~ 5. 7 J- M. CRla;F,'rn .,\XJ) B. lkL I(t':CORD, lli:)chem. ,/., 52 {I()52 ) 23(). 8 F. I'A'rAT ..\XD I-I. (.;. I';LI,XS, .Vatit;'zlis.s:'nschaf/e~*, .t0 ;I959} 322. 9 't'. C. I_,Av:,O.cx'r, Arch. Biochem. l)iopi!ys., 9"-' (190I) 22.I-
io S. [,. |"}uv.sox, JR., M. J. FAHRISNBACH, g. [[. i"ROM~IIt.kGEN, B. :\. RIO.AInu, R. A. }3ROWX, J . . \ . BI<ov:,cx*axx, H. V. lmwl<v ~xx> E. L. R. S'roKs'ra,~, . / . . 4m . Chem. So~., 78 (I9.56) 587.!.
I I J. [<. III':I,BERT AND M. A. ~I.".RINI, Biochemistry, 2 (19r53) II~or. r2 S. [". LASI-2ER A.N'I) S. S. ,qTIV.XI,A, Arch. Bi:,chem. 13iophys., I t 5 (I966', 36c;. 13 K. I-1..~[I.;VER AND I). [".. SClIW.\RrZ, He:e:. Chim. Acla, 33 (105 °) I65: . 14 A..B. Fosr~.'R, ~r. 5c:xc~,Y, 1". J..M. "l',\vt.oe, J. 31. \VLI-m.;R ..\ND M. L. WOLF~eOSL Biochcm../ .
80 (19GII I3P. .I 5 S. E. J,~\SK~ZR, i'lL 1). l)isscrt;~tion, S tevens In s t i t u t e of "l'e~hno ,'~gy, 19(~5, No. 65-~2 579
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