BIOCHIMICA ET BIOPttYSICA ACTA 141

BBA 85012

T H E I N T E R A C T I O N B E T W E E N P O L Y S A C C H A R I D E S

A N D O T H E R M A C R O M O L E C U L E S

VII . T H E EFFECT OF VARIOUS POLYMERS ON T H E SEDIMENTATION

RATES OF SERUM ALBUMIN AND a-CRYSTALLIN

TORVARD C. LAURENT AND HAKAN PERSSON

Department of Medical Chemistry, University of Uppsala, Uppsala (Sweden)

(Received September ioth, 1963)

SUMMARY

The sedimentation behaviour of two globular proteins of different size has been studied in solutions of polymers of varied structure. That the movement of the larger protein was in all instances retarded to a greater extent than tha t of the smaller indicated that the polymers investigated had a sieving effect. The ability of a polymer to inhibit the migration of another substance was demonstrated to increase with its degree of polymerization and decrease with branching. Charged substituents in a polymer were found to augment materially its effectiveness in restricting transport.

INTRODUCTION

I t has been shown that the presence of hyaluronic acid decreases the sedimentation and diffusion rates of other substances1, 2 and that this effect can be regarded as a macromolecular sieve action. The extent to which migration is inhibited is basically a function of the concentration of the polysaccharide and the size of the moving particle S. The sieve effect of hyaluronic acid is of such nature as even to permit its utilization in increasing resolution in the analytical ultracentrifugO.

The present paper describes the influence of a number of polymers, mostly poly- saccharides, on the sedimentation rates of two substances, ~-crystallin and human serum albumin.

An endeavour has been made to evaluate the importance of the internal structural features of a polymer, which may contribute to its ability to regulate transport. Molecular weight, degree of branching and charge have been emphasized in the in- vestigation.

MATERIALS

The same preparations of bovine ~-crystallin and human serum albumin as described in previous reports 2, ~ were used.

Dextrans, ficoll and dextran sulphate (sodium salt) were obtained from AB

Biochim. Biophys. Acta, 83 (1964) 141-147

/

i4~ T.C. LAURENT, H. PERSSON

Pharmacia, Uppsala, Sweden. The dextrans had molecular weights of lO 4, 8.1o 4, 5" lO5, 2" IO e and 2.5" lO7-5 • lO 7. Approx. 94 % of the glucosidic bonds in the dextran preparations were in 1,6-position. The ficoll, a compact, highly branched synthetic polysucrose had an average molecular weight of lO 6. The dextran sulphate was synthe- sized from a dextran with a molecular weight of 2. lO s. I ts sulphur content was 17 ~: 0.5 %. In sedimentation experiments it showed a small slower component in addition to the main component. Solutions of known concentrations of dextran, ficoll and dextran sulphate were prepared in boiling water. The absolute concentrations of dextran sulphate solutions were multiplied by 0.45 to obtain the concentrations of unsubstituted polymer.

Methylcellulose was obtained from L. Light and Co. Ltd, Colnbrook, England. I t had a limiting viscosity number of 350 ml/g, which corresponds to an approximate molecular weight of 8. lO 4 (ref. 4). The methylcellulose powder was wetted with a portion of the solvent heated to 8o°-9 o°, before it was dissolved at room temperature with constant stirring. The stock solution was stored at 4 ° until used. To obtain the concentrations of unsubstituted polymer, methylcellulose concentrations have been multiplied by 0.89, a factor which corresponds to an assumed degree of substitution of 1.5.

CM-cellulose (Cekol HVB) was obtained from Uddeholms AB, Sweden. I t was used as a sodium salt. The preparation was suspended in solvent, vigorously stirred for 30 rain, and then centrifuged at 60000 × g for 60 min to remove undissolved gel particles. The concentration of the supernatant was determined in a Spinco analytical ultracentrifuge by layering solvent over the solution in a synthetic boundary cell and measuring the area under the schlieren peak. The correspondence between area and concentration was found by previous calibration with a solution of a com- pletely soluble low molecular weight CM-cellulose of known concentration. The re- fractive increment of the polysaccharide was calculated to be o.139 ml/g. The limiting viscosity number of the preparation was 41o ml/g in 5 % NaOH. The degree of substi- tution was taken to be o.61 on the basis of two independent methods of estimation: (a) Ashing of the polymer and titration with o.I N sulphuric acid. (b) Titration of carboxymethyl groups with cetylpyridinium chloride 5. The concentration of un- substituted polymer was obtained from its actual concentration by multiplication with the factor 0.83.

Polyvinylalcohols ("Polyvior ') were obtained from Firma Wacker Chemie, GMBH, Munich, Germany. The four samples used, W 05/20, W 13/2o, W 28/20 and W 48/20 had molecular weights of 22. lO 8, 44" lO3, 7 °. lO3-75 . lO3 and lO 5 according to the manufacturer. The polymer was brought into solution at ioo °.

Chondroitin 4-sulphate (sodium salt) was prepared from bovine nasal septum in the manner described by SCOTT 6. I ts nitrogen and sulphur contents were 2.89 and 6.61, respectively. The limiting viscosity number of 54 ml/g and sedimentation constant of 1.73.IO -lz sec correspond to an approximate molecular weight of 15" IO~-2o "lob (refs. 7, 8).

Corrections were applied for the moisture content of the polymers, when solutions of specific concentrations were made up directly from solid preparations. The moisture content was determined by drying the samples over phosphorus pentoxide at IiO °. ~-Crystallin and serum albumin were used in concentrations of o.oi g/ml. The medium employed in all experiments was 0.2 M NaCI.

Biochim. Biophys. Acta, 83 (1964) 141-147

THE SIEVE EFFECT OF VARIOUS POLYMERS 143

METHODS

Sed imenta t ion rates were de te rmined in a Spinco Model E ana ly t i ca l u l t racent r i fuge as descr ibed earlier1, 2. Wheneve r the sed imenta t ion ra te of the po lymer exceeded t ha t of the prote in , syn the t ic pro te in boundar ies were c rea ted b y using va lve - type syn the t ic b o u n d a r y cells. Viscosities were de te rmined at 20 ° in an Os twald v iscometer wi th an outflow t ime for wate r of I iO sec.

RESULTS

Two sets of exper iments were per formed to s t u d y the impor t ance of the molecular weight of a po lymer on i ts ab i l i ty to r e ta rd the m o v e m e n t of a globular par t ic le .

'%~ /.s 15 * . /

3 d "

3 ~-~.~.-..~ ' ~ . • ~ Z - - - - ---=~'~-: . . . . . . . 1

~ 1 ~ ~----_~_-~--~ - - - ~ ~ I I i I 0 1 2 3

Concentration of Dextran (g/rn[ u 10 2)

Fig. i. The sedimentation rate of c¢-crystallin ( --) in the presence of (i) dextran with molecular weight of lO4; (2) 8"1o4; (3) 5"IO~; (4) 2.IO ~ and (5) 2.5"IO7-5 "Io ~. ( . . . . ), the sedimentation rate of dextran measured in separate runs; ( - . - - . - ) , the relative viscosity

of the polymer solutions (~reL).

9 / / "

--~o / /

$ 4 • ./

./~_~.~.. ~ - ~

o 1 2

Concentration of Polyvinylatcohol(g/rnMO~

Fig. 2.The sedimentation rate of serum albumin ( ) in the presence of (I) polyvinyl alcohol with molecular weight of 22.1o3; (2) 44"IO~; (3) 7o'IoL75"IO~ and (4) lO5- ( . . . . ), the sedimentation rate of polyvinylalcohol meas- ured in separate runs; ( . . . . . ), the relative

viscosity of the polymer solutions (?]rel.)-

Fig. I i l lus t ra tes the effect of a series of dex t rans on the sed imenta t ion ra te of ~- crysta l l in , and Fig. 2 the influence of var ious po lyv iny la lcohols on the sed imenta t ion of serum albumin. The same graphs also show the re la t ive viscosit ies of the po lymer solut ions as well as the sed imen ta t ion rates of the ind iv idua l po lymers measured in separa te runs.

Three neu t r a l po lymers wi th va ry ing degree of branching , ficoll, dex t ran and methylcel lulose , are compared in Fig. 3, where the i r influence on the t r anspo r t of =-crystal l in and a lbumin are shown. Here the concent ra t ion of po lymer has been expressed on the basis of the pa ren t unsubs t i t u t ed polysacchar ide . In the case of methylcel lulose, for example , the me thy lg roup concent ra t ion has been subs t r ac t ed from tha t of the cellulose der iva t ive ac tua l ly employed. The polysacchar ides invest i - ga ted r e t a rded the sed imen ta t ion of prote ins in order of increasing chain l inear i ty : ficoll < dex t r an < methylcel lulose.

Biochim. Biophys. dcta, 83 (1964) 141-147

144 T . C . LAURENT, H. PERSSON

15 \ ~ the i

• / 20

~o ~° 3,, / - ~ .~,-.

'~ '~. .~ ~ e e ~ o 2 3 ~ , ~ ~ . 3

l I I I I I 2 3

Concentr(~tion of Onsubstituted Polymer ( g / ~ t . ~o 2)

Fig. 3. The s ed imen ta t i on ra te of ~-crysta l l in (top) and s e rum a l b u m i n (bot tom) ( ) in t he presence of (I) ficoll; (2) d e x t r a n (mol. wt. 2.zoO); a n d (3) methylcel lulose . ( . . . . ), t he s ed imen ta t i on ra tes of t he po lymers measu red in sepa ra te r uns ; ( . . . . . ), t he re la t ive v iscos i ty

of t he p o l y m e r so lu t ions (~rel.).

15 ~ /2 r/re I

10 10"

~2

"~'~.'-~2 l " < t ~ - °

Concen t ra t i on of Unsubs t i tu ted Potymer~ (g/rot,)0")

Fig. 4. The s ed imen ta t i on ra te of ~¢-crystallin (top) and s e rum a l b u m i n (bot tom) ( ) in the presence of (i) d e x t r a n (mol. wt. 2- IO s) and (2) d e x t r a n su lphate . ( . . . . ), t he sed imen- t a t ion ra tes of t he po lymers m e a s u r e d in sepa- ra te runs ; ( . . . . . ), t h e re la t ive viscosit ies of

t he po lymer solut ions (~re0.

I=" ~/~ / "r] rel

30

10 2./ / 1 25

/ . / 20"

" " 15

- / / . . % • • 2

2

1L "~. 2* e~,l I , - . - - - ; - - 7 - ,

0 1 2 3 Concentration of Unsubstituted Polymer

( g / m L , lo 2) Fig. 5. The s e d i m e n t a t i o n ra tes of ~-crystal l in (top) and s e rum a l b u m i n (bottom) ( ) in the presence of (I) methylce l lu lose and (2) CM- cellulose. ( . . . . ), t he s ed i men t a t i on ra tes of t he p o l y m e r s m e a s u r e d in sepa ra te runs ; ( . . . . . ), t h e re la t ive viscosit ies of t he po l ymer

solut ions (~hel.).

S" 10( No

07E

1150

0.25

0

0.75

0.5O

02E

"<"B'~ K-- B "~ D

; I I I I

1 2 3 4 1 2 3 4~ Concentration of Po[ymer (g/m1,10 z)

Fig. 6. The rela t ive decrease in s e d i m e n t a t i o n ra te of s e r u m a l b u m i n ( • - - • ) and ~-crysta l l in ( × - - × ) in the presence of (A) ficoll; (t3) dex- t r an (mol. wt. 2 . ioe) ; (C) d e x t r a n su lpha te ; (D) CM-cellulose; (E) methylce l lu lose ; (F) poly- v inyl alcohol (mol. wt. lO 5) ; and (G) chondroi t in 4-sulphate . S is the s ed imen ta t i on ra te in t he

presence, S o in the absence of polymer .

Biochim. Biophys. Acla, 83 (1964) 141-147

THE SIEVE EFFECT OF VARIOUS POLYMERS 145

The effects of dextran and dextran sulphate have been contrasted in Fig. 4, and of methylcellulose and CM-cellulose in Fig. 5. The concentration of polysaccharide has again been expressed on an unsubstituted basis. In both series of experiments, the charged polymer was found to restrict protein movement markedly more than its neutral analog.

The inhibitory capabilities of the various polymers are shown in Fig. 6 in terms of the relative decrease in the sedimentation rates of albumin and a-crystallin. In all instances, the larger molecule, a-crystallin, was retarded to a greater degree than the smaller albumin.

DISCUSSION

The observation that a polysaccharide in solution can restrict the transport of other substances and in a manner that is dependent on the size of the transported particles 1, 2 not only has important implications in biological systems, but also suggests the possibility of practical applications of the phenomenon in various preparative and analytical methods 3. A detailed investigation of those polymer properties, which were most likely to be essential for the manifestations of a polymer 's sieving action was therefore undertaken.

A number of polymers have been studied and in all cases they have retarded the sedimentation of the two proteins used as test substances. The retardation of the larger molecule, c,-crystallin (mol. wt. 83oooo) has always proved to be greater than that of serum albumin (mol. wt. 67 ooo) (Fig. 6) in agreement with the hypothesis that the polymers in the media employed functioned as sieves.

I t was shown earlier 1 that of two hyaluronic acids with different degree of poly- merization, the higher molecular weight polysaccharide retarded the sedimentation of albumin to the largest extent. The experiments with dextran (Fig. I) and polyvinyl- alcohol (Fig. 2) are in accord with the previous study. The variation in the transport inhibitory capability of dextran with its degree of polymerization was most marked in the lower molecular weight range (Fig. I) while the difference between dextrans of molecular weights 5" IO5 and 2.5" lO7-5 • IO ~ was relatively small.

The relative viscosities of the polymer solutions have been plotted in Figs. I -5 to show that their retarding effect on the globular particles is not directly correlated to the viscosity. From Fig. I, one can calculate that in 3" IO-Z g/ml dextran, the sedimentation rate of a-crystallin in solutions of polymers with molecular weights of lO 4 and 2.5" IO7-5 • IO I will be, respectively, 0.69 and 0.27 of the original, whereas the corresponding reciprocal relative viscosities of these media are 0.77 and o.14. Similarly, the lowest and highest molecular weight polyvinylalcohols decrease the sedimentation rate of albumin to 0.42 and 0.34 of its original value, while the corre- sponding reciprocal relative viscosities are 0.45 and o.17. The discrepancy between the extent to which the sedimentation of a substance is decreased and the relative viscosity of the polymer solution in which this occurs, seems to be largest for the linear polyelectrolytes. CM-cellulose in concentrations of 0.005 and o.oi g/ml and with relative reciprocal viscosities of o.18 and 0.08 reduced the sedimentation rate of ~-crystallin to 0.50 and o.31 of that of the pure protein.

Ficoll, dextran and methylcellulose were compared to evaluate the influence of branching within a polymer molecule on its capacity to retard the migration of

Biochim. Biophys. Acta, 83 (1964) I 4 I - I 4 7

146 T. C. LAURENT, H. PERSSON

proteins. The first two polymers had molecular weights of the same order of magni- tude, but the latter had a much lower degree of polymerization. Despite this, the effect produced by methylcellulose was far larger than that of the others (Fig. 3). These experiments clearly show that an increase in branching materially reduces a polymer's ability to affect the sedimentation of globular proteins. In the foregoing as well as in the experiments summarized in Figs. 4 and 5, the concentration of polymer has been expressed on an unsubstituted basis, i.e. in terms of the concen- tration corresponding to a certain total chain length per unit volume in the solution. I t was thought that this way of expressing the concentration facilitated comparison. If absolute concentrations had been used instead, the changes in Fig. 3 would in any event have been too small to invalidate the conclusions made.

Figs. 3 and 4 show that even though dextran and dextran sulphate sediment faster than albumin, the sedimentation rate of albumin actually decreased in their presence. I t should be pointed out that the sedimentation rates of the polysaccharides were measured for solutions of pure polymer in separate runs, and due to the John- ston-Ogston effect, the sedimentation rate of polymers in a mixture is not the same. The results, nevertheless, indicate that the retardation of the protein must be a complex phenomenon that is not solely due to a lowering of a particle's ability to penetrate a unit of volume in its path because of the simple intervention of a poly- saccharide network. Other factors, which may have a role and require consideration are the increased density of the polymer solution over that of the solvent and the possibility of density fluctuations in the solution arising from the sterical exclusion of protein molecules from the domain of the dextran molecule 9.

The effects of charged and uncharged polymers have been compared in Figs. 4 and 5, where it can be seen that charged polymers retard the sedimentation of proteins to a greater extent than do neutral polymers. The difference between dextran and dextran sulphate is larger than that between methylcellulose and CM-cellulose, probably because of the higher charge density on dextran sulphate than on CM- cellulose (1. 9 and 0.6 negative charges per monosaccharide, respectively).

The fact a charged polymer appears more able to retard a globular particle than a neutral one, seems at first to be contradictory to an earlier observation 1 that variation of pH between 5-5 and 8.5 and ionic strength between 0.02 and 0.5 does not alter the effect of hyaluronic acid on the sedimentation rate of albumin. One must conclude that contribution of charge to the increased effectiveness of a polymer in regulating transport involves more than merely charge interaction between the polymer and the sedimenting particle. An expansion of the polymer chain stemming from repulsive forces between neighbouring charges should increase the effective length of the polymer chain and make it more rigid. The consequences of this can be envisaged as advantageous if the sieve effect is caused at least in part by the mechanical hindrance encountered by particles in a three-dimensional network of polymer chains.

The conclusions drawn from this investigation are that high molecular weight, charged linear polyelectrolytes are most effective in retarding the movements of other macromolecules. That hyaluronic acid, an important constituent of connective tissue, is such a molecule may be of great biological significance.

Biochim. Biophys. Acta, 83 (1964) 141-147

THE SIEVE EFFECT OF VARIOUS POLYMERS 147

ACKNOWLEDGEMENTS

The authors wish to acknowledge gifts of various research samples from Dr. K. GRANATH, AB Pharmacia; Mr. H. BJ(~RLING, AB Kabi and Uddeholms AB. Mr, A. PIETRUSZKIEWICZ kindly corrected the manuscript. The investigation was supported by the Swedish Medical Research Council and the Swedish Cancer Society.

REFERENCES

1 T. C. LAUREI~T AI~D A. PIETHUSZHIEWlCZ, Biochim. Biophys. Acta, 49 (1961) 258. 2 T. C. LAURENT, I. BJ6RK, A. PIETRUSZHIEWICZ AND H. PERSSON, Biochim. Biophys. Acta, 78

(1963) 351 • 3 T. C. LAURENT AND H. PERSSON, Biochim. Biophys. Acta, 78 (1963) 360. 4 G. K. GREMINGER, Jr . AND A. ]3. SAVAGE, in R. L. WHISTLER AND J. N. BEMILLER, Industrial

Gums, Academic Press , New York, 1959, p. 565 • 5 j . E. SCOTT, Chem. and Ind. London, (1955) 168. 6 j . E. SCOTT, in D. GLICK, Methods o/Biochemical Analysis, In te r sc ience Publ . , N e w York, 8

(196o) 145. 7 M. B. MATHEWS AND A. DORFMAN, Arch. Biochem. Biophys., 42 (1953) 41. 8 T. C. LAURENT AND A. ANSETH, Exptl. Eye Res., I (1961) 99. 9 T. C. LAURENT, Biochem. J., 89 (1963) 253.

Biochim. Biophys. Acta, 83 (1964) 141-147