Biochimica et Biophystca Acta, 427 (1976) 197-207
© Elsevier ScientificPublishing Company, Amsterdam- Printed in The Netherlands BBA 37260 POLYMERIZATION OF PROTAMINE SULPHATE BY CARBODIIMIDE AND INTERACTION OF ISOLATED PROTAMINE POLYMERS WITH H U M A N RED BLOOD CELLS
N. M. BARFOD and B. LARSEN The Institute of Cancer Research, Radiumstationen, DK-8000 Aarhus C (Denmark)
(Received July 22nd, 1975)
SUMMARY A method using a water-soluble carbodiimide to polymerize protamine sulphate is described. The behaviour of polymerized protamine in Sephadex chromatography and in polyacrylamide gel electrophoresis indicates that protamine has been polymerized into aggregates with defined molecular weights. Turbidimetrical titrations of the isolated protamine polymers with dextran sulphate show that the cationic charge density has been conserved after polymerization. The binding characteristics of the protamine polymers to human red blood cells as measured by cell electrophoresis indicate increased affinity with increased molecular weight of the polymer.
INTRODUCTION It is a well-documented fact that the molecular size of proteins and other macromolecules plays an important role in their biological interactions. Evidence has accumulated of the importance of this molecular parameter in the interaction of cell surface-binding macromolecules such as lectins, antigens and polycations [1, 2]. Thus, cross-linking of soybean agglutinin into high molecular weight complexes with glutaraldehyde caused a 100-200-fold higher specific haemagglutinating activity than that of the native lectin when tested with human erythrocytes [1]. Maximum stimulation of [3H]thymidine incorporation into mouse spleen lymphocytes by cross-linked soybean agglutinin was obtained at a concentration that was five times lower than that required for maximum stimulation by the unmodified lectin [1]. Furthermore, concerning the mitogenic activity of two haemagglutinating compounds (a dimer and a tetramer) purified from the Lima bean (Phaseolus lunatus), the tetramer was several times more active than the dimer form [2]. Reichart et al. [3] found that acetylated concanavalin A, a dimer, possessed about one half the potency of tetrameric concanavalin A as a mitogen for human lymphocytes. The antibody response in guinea pigs and rabbits demonstrated that glutaraldehyde-polymerized ragweed-pollen antigen E exhibited greater antigenicity than the monomeric ragweed antigen E [4]. Finally, the polycation-enhanced uptake of albumin into tumour cells showed increased effect with increasing molecular weight (Mr) of polycations [5], and
198 in parallel with this it was found that the tumour-toxic effect was increased by increasing molecular weight of the polycation [6, 7]. In the elucidation of the significance of molecular size of proteins for interaction chemical polymerization may be employed. Generally, polymerization of proteins is difficult to control since proteins are rather complex molecules with many reactive side groups capable of a multitude of reactions. Recently, we showed that the low molecular weight basic protein protamine from Salmo irideus (iridine) could be polymerized by a water-soluble carbodiimide into defined aggregates, which were separated by Sephadex chromatography [7]. The tumour-toxic effect on JB-1 ascites tumour cells of the partially separated protamine polymers showed increased effect with increasing molecular weight of the aggregates. Finally, it was shown that the increased tumour-toxic effect of the high molecular weight polymers could be correlated with increased affinity for the tumour cell surface as measured by cell electrophoresis. Here we describe an improved method for polymerization of the polycation protamine (iridine) with carbodiimide giving rise to dimers, tetramers, hexamers, etc. Fractionation of the polymers and some preliminary chemical characterizations are also reported. Since polycations bind to the anionic sites on the cell surface, the purified aggregates were tested by their affinity for human red blood cell surfaces as measured by cell electrophoresis. MATERIALS AND METHODS
Polymerization procedure. Protamine sulphate (Nordisk Insulin Laboratorium), 1.0 g, was dissolved in 30 ml of glass-distilled water and cooled to 0 °C. A water-soluble carbodiimide, 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide, (Sigma Chemical Co.), 10.0 g, in 20 ml of glass-distilled water (0 °C) was slowly added. The solution was adjusted to pH 5.0 with 1.0 M HC1. The mixture was allowed to react for 6-10 days in a refrigerator (4 °C). Thin-layer gel filtration. Thin-layer gel filtration was performed with the Pharmacia thin-layer gel filtration apparatus on Sephadex G-75 superfine. The gel layer was allowed to equilibrate for 18-20 h before each run. After completion of the run the sample substances in the gel were transferred to Whatman 3MM filter paper stained with 0.1 ~ Bromophenol blue in water/acetic acid (95:5, v/v) saturated with HgC12 and rinsed in water/acetic acid (95:5, v/v). Molecular weight estimations. Molecular weights of protamine and polymers of protamine were determined by thin-layer gel filtration by developing them together with reference proteins (Mr in parentheses): cytochrome c (12400), myoglobin (17 800), chymotrypsinogen A (25 000), ovalbumin (45 000), bovine serum albumin monomer (67 000), bovine serum albumin dimer (134 000) (Schwarz/Mann, Orangeburg, N.Y.). Reference lines were obtained by plotting the inverse migration distances relative to Blue dextran (2 000 000) against the logarithms of the molecular weights. Column chromatography. The reaction mixture was fractionated on a column (5.0 × 90 cm) packed with Sephadex G-75 superfine. Fractions were monitored by the method of Lowry et al. [8] at 750 nm. All gel filtration procedures including thinlayer gel filtration were performed with 0.15 M NaC1 as eluent. Pooled fractions were dialysed against distilled cold water (4 °C) and lyophilized.
199
Turbidimetric titrations. The charge density of protamine and protamine polymers was compared by titration with the polyanion dextran sulphate (containing 17 :k 0.5 ~o S and 6.5 ~ water). Generally, cationic and anionic polymers react stoichiometrically in diluted solutions [9, 10]. The titration is followed by an increase in turbidity caused by the formation of colloidal polycation-polyanion complexes until the electrochemical point of equivalence is reached. Addition of more polyanion causes a decrease in turbidity owing to precipitation of insoluble complexes. Turbidity was measured at 340 nm after 20 min. Gel electrophoresis. Polyacrylamide gel electrophoresis was performed by the method of Panyim and Chalkley [11]. The gels were stained overnight in 0.1 ~o Coomassie brilliant blue dissolved in 7 ~ acetic acid and destained by diffusion against 7 ~o acetic acid. Cell electrophoresis. Preparation of normal human erythrocyte suspensions and measurements of cellular electrophoretic mobilities were performed as recently described [7]. RESULTS
Chromatography The degree of polymerization resulting from different conditions for the reaction was determined by thin-layer gel filtration in order to trace the optimal circumstances for preparative purposes of polymerized protamine. It was found that for optimal polymerization the reaction requires (l) low temperature, (2) pH 4-5, (3) high concentrations of protamine and carbodiimide, and (4) a reaction time of at least 3 days. Stirring was avoided because this ruined polymerization. Preparative fractionation on Sephadex G-75 superfine yielded three peaks as seen in Fig. 1. The reason why the eluate was investigated by the Lowry method of protein analysis was that protamine as well as carbodiimide were stained by this procedure. The large slow eluting peak is carbodiimide. After dialysing and lyophilizing
2.0
1.5
1.0
! 0,5
I 0 40
60
80
100
120
Fraction number
Fig. 1. Column fractionation of carbodiimide-polymerized protamine on Sephadex G-75 superfine.
200 of the three pooled polymer peaks the fractions were rechromatographed on thinlayer gel filtration. It was found by thin-layer gel filtration that only partial isolation of the polymer classes was achieved. Rechromatography of the highest molecular weight fractions was performed on G-100 superfine Sephadex, and of the more low molecular weight fractions on G-50 superfine. Thin-layer gel filtration of the isolated
!
!
! 0
i,
Startla~ Im Fig. 2. Thin-layer gel filtration of isolated protamine-polymerspecies from carbodiimide polymerization.
fractions yielded nearly complete separation of protamines with homogeneous molecular weights (Fig. 2). Molecular weight estimations based on thin-layer gel filtration showed uniform protamines with approximate molecular weights of 6000, 11 500, 24 500 and 39 000. Judging from these data, we presume that protamine of molecular weight 6000 is polymerized quantitatively into dimers (Mr 11 500), which may condense into tetramers and hexamers. Since we have only six standards there is considerable uncertainty in estimation of molecular weights of polymerized protamine so that these values must be taken as rough approximations.
201 Titrations
Accurate protein analysis of protamine and the isolated polymerizates had to be performed with the Biuret reagent. The Lowry method was not employed because (1) it lacked linearity of standard curves and (2) it stained carbodiimide and might thus interfere with the protein analysis. Carbodiimide did not stain with the Biuret reagent. Amounts of 3.0 ml of 0.1 mg/ml (determined from a protamine Biuret standard curve) of protamine monomer, dimer, tetramer and hexamer were turbidimetrically titrated with 1.0 mg/ml dextran sulphate. The result of the titrations appears from Fig. 3. The almost identical point of equivalence of the titration curves indicates that none of the cationic groups of protamine was lost during the polymerization process. Thus, the only variable molecular parameter of the protamines was the molecular weight. It is seen from Fig. 3 that the titration curve for the higher molecular weight species ascends more rapidly before maximal turbidity, and descends more rapidly beyond the maximum. This may be caused by a higher affinity of the high J
0,60
i
0.50 f
0A0 I i E ¢
0.30,-
0.20 i
di-PS
I i
tetra-PS
0,10 /
hexa-PS
0
50
100
150
200
250
300
ILl 1.0mg/ml Dextran Sulphate
Fig. 3. Turbidimetrical titration of protamine sulphate (PS) and isolated protamine polymers (diPS, tetra-PS, etc.) with dextran sulphate.
202
Fig. 4. Polyacrylamidegel electrophoresis at pH 3.2 in 8 M urea. Right gzl, protarnine; left gel, carbodiimide-polymerizedprotamine.
molecular weight material for dextran sulphate: When dextran sulphate is added to the protamine solution, the high molecular weight species react more readily with dextran sulphate giving more turbidity. Beyond the point of equivalence, the dextran sulphate-protamine polymer complexes precipitate more rapidly because of larger dimensions of the polyanion-polycation complexes. Analysis of the amount of arginine [12], the only amino acid of protamine containing a charged group, yielded 83 g arginine per 100 g protamine base, which gives 28.2 mol of arginine per mol of protamine base (assuming a molecular weight of 6000 of protamine base). Calculations concerning the titration of protamine with dextran sulphate revealed that negatively charged sulphate-esterified groups in dextran sulphate react nearly stoichiometrically with the positively charged arginyl residues in protamine (1.00 #mol arginyl residues were titrated with 1.08/~mol of S), and that no cationic groups were lost during polymerization.
Gel electrophoresis Polyacrylamide gel electrophoresis at pH 3.2 under denaturing conditions in 8 M urea showed that the aggregates were covalently coupled (Fig. 4). From the bands in the upper part of the gel obtained after electrophoresis it is seen that the reaction goes on to produce polymer aggregates with molecular weights beyond the
203
1.0
0.5
0.4
'~ 0.3
0.2
0.1
6 000
12 CO0
24 000
36 000
48 000
60 000
Molecular weightsoipolymerized protamine
Fig. 5. Ferguson plot of mobility versus the expected molecular weights of polymerized protamine (dimers, tetramers, etc.). protamine hexamer. However, it was not possible to isolate detectable amounts of such higher molecular weight protamine polymers. Since the cationic charge density of the protamine polymers is identical as determined from the titration curves, the retardation coefficient KR may be assumed to be proportional to molecular weight of the different polymers. Thus, a linear relationship should exist between the logarithm of the electrophoretical mobility and molecular weight according to Ferguson [13]. Assuming that monomer protamine (Mr 6000) is polymerized into dimers ( M r 12 000), tetramers ( M r 24 000), hexamers (M~ 36 000), octamers (M~ 48 000), and decamers (Mr 60 000), a plot of molecular weight versus log mobility should represent a straight line. This is in fact the case as seen from Fig. 5, although some deviations are observed for monomer and dimer protamine.
Cell electrophoresis As a consequence of high negative cell surface charge [4], red blood cell surfaces bind a variety of positively charged compounds, including protamine and other polycations. The adsorption of polycation on the cell surface is accompanied by a reduction of the negative surface potentials, and even positive values of the surface potential may be obtained with increased concentrations of polycation [6]. Consequently, cell electrophoresis was a convenient technique for studying the binding of protamine polymers to human red blood cells. The electrophoretical mobility (M v/E, where v ~ velocity of the cell in the electrical field and E = field strength) was plotted against the logarithm of the concentration of added polycation as seen in
204
hexa-PS +0,5
"7 :> '7 0.0
/
~
=. =E
-0.5
1
-1.0
Fig. 6. Effect of protamine and protamine polymers on electrophoretical mobility of human red blood cells. Temperature, 25 °C. Measured in phosphate-buffered saline/Sorbitol buffer (1:4, v/v); ionic strength, 0.035; cell concentration 5.105/ml. S.D. for each measurement is shown in the figure. Mobility determinations are uncertain when approaching zero, which is indicated with brackets. Fig. 6. It is obvious that the binding of protamine to the cells was enhanced with increasing molecular weight of protamine since smaller amounts of high molecular weight protamine were required to neutralize the negative surface potential. A relative expression of the affÉnity of the various polymers for red blood cells may be obtained by comparing the amount of polycation necessary to furnish the cell with a given mobility. Since the surface charge should be identical for cells with equal mobility, it is assumed that the number of positive groups adsorbed per cell must be identical for the various polymers in order to obtain a given mobility. The amount of the various polymers which have to be added to give zero mobility of the cells represents the sum of the concentrations of the cell bound polycation, Cbound, and the unbound, free polycation, Cfre~. At zero surface potential the amount of polycation bound to red blood cell surfaces is less than 0.1 #g/ml for l0 7 cells/ml [17]. The total added amounts, the values of which are found from the intersection of the curves with the abscissa (at zero mobility) in Fig. 6, may for practical purposes replace Cfre~ since the value of Cbou,d is negligible. The association constant, Kas ~
Cbound Crree × (available binding sites)
may be replaced by the "relative affinity", 1/Cfree as the concentration of red blood cells, i.e. the number of available binding sites is kept constant. Taking the relative affinity of monomer protamine for red blood cells as unity
205
Mw
40 000
30 000
20 000
10 000
0
I
I
L
i
1
5
10
20
Relative Affinity
Fig. 7. The relation between molecular weight of protamine and relative affinity for human red blood cells.
and the affinities of the polymers relative to that, a plot of molecular weights of the different protamines against the logarithm of the relative affinities represents a straight line as seen in Fig. 7. This type of relation between molecular weight and association constants by interaction points to cooperativity in the binding reaction. If S is a cooperativity constant and Ka"ss is the association constant of the n polymer, the following relation exists K~,~ = S ("- 1) K~s where Ka~s, is the m o n o m e r association constant. This bears resemblance to the properties of the interaction mechanism depicted in Fig. 7. DISCUSSION Generally, carbodiimides react with carboxyl groups at slightly acidic p H levels giving an acylisourea, an activated intermediate that in turn can either rearrange into an acylurea or react with a nucleophile [15]. In the present study we used protamine (iridine) both as carboxyl donor for carbodiimide and as a nucleophile. F r o m a theoretical point of view it is likely that these conditions should lead to peptide-bond formation between the protamine molecules. Since the amino acid composition of iridine, in addition to arginine, consists of only neutral amino acids such as proline,
206 serine, valine and glycine, the only possible point of action for carbodiimide is the N-terminal amino group and the C-terminal carboxyl group. This would account for end-to-end polymerization giving rise to protamine polymers with molecular weights of n x 6000. As already mentioned we find only dimers, tetramers, hexamers, etc., not trimers, nor pentamers. These molecular weights were estimated by thin-layer gel filtration and the assumption of dimerization, tetramerization, etc. was supported by a Ferguson plot (Fig. 5). An explanation might be that protamine is first converted quantitatively into dimers, which subsequently are able to condense into tetramers, hexamers and octamers. The possible linearity of the protamine polymers as a consequence of end-toend coupling could be proved by theoretical calculations of actual measurements of polymer flexibility and shape in solution for dimer, tetramer and hexamer protamine. However, since physicochemical measurements may be more or less subject to interpretations, a more direct proof would be chemical analysis of the amino acid sequences in peptides obtained from monomer and polymerized protamine after protease (e.g. trypsin) digestion. Experiments are under way to test this lastmentioned approach. Polymerization with carbodiimide was found to be superior to formaldehyde or glutaraldehyde polymerization when dealing with basic proteins such as protamine. We found a decreased charge density of protamine after glutaraldehyde polymerization, but the charge density of carbodiimide-polymerized protamine was unchanged as estimated from the titration curves (Fig. 3), and the binding to red blood cells was greatly increased after polymerization. Ryser [5] demonstrated in 1967 that the biological activity of polycations increases with the molecular weight, which was shown with homologous series of polycations as polylysin and DEAE-dextrans with varying molecular weight. A linear correlation was found between the log molecular weights and log albumin uptake into Sarcoma S 180 II mouse tumour cells. It was claimed that the molecular mechanisms underlying this statement were that an interaction of the polymers with the cell membrane was increased in effectiveness by the number of simultaneous attachments to the membrane [5, 9]. The fact that the molecular weight of protamine polymers is proportional to the logarithm of the relative affinity as seen in Fig. 7 points to the presence of cooperativity in this type of interaction [16]. The cooperativity of interaction is characterized by the influence on the binding constant of one group of the polymer to a binding site by the previously bound group of the polymer; K~"ss= S c " - 1 ) K,st s. The similarity of this relation between molecular size and affinity of the binding of polycations to red blood cell surfaces with the experimental data depicted in Fig. 7 may be suggestive for the assumption that cooperative forces are operating. As a result of the formation of such very strong binding forces conformational alterations of the cell membrane are apt to occur, both in the molecular and the structural dimensions, leading to alterations of cellular activity as previously shown [7]. To recapitulate, we have described a method using a water-soluble carbodiimide to polymerize protamine into aggregates with defined molecular weights. Furthermore, we have found a conservation of the cationic charge density of the isolated protamine polymers, which was confirmed by measuring the relative affinity of protamine polymers for the human red blood cell surface. In this manner we have
207 shown that cooperativity may play an i m p o r t a n t role in the interactions of polycations with cell surfaces. ACKNOWLEDGEMENTS We wish to t h a n k Mrs. Jytte Olsen a n d Miss Pia Ohl for the p r e p a r a t i o n of the figures a n d N o r d i s k I n s u l i n L a b o r a t o r i u m for the supply of p r o t a m i n e sulphate. This work was sponsored by the D a n i s h Cancer Society. REFERENCES 1 Lotan, R., Lis, H., Rosenwasser, A., Novogrodsky, A. and Sharon, N. (1973) Biochem. Biophys. Res. Commun. 55, 1347 2 Ruddon, R. W., Weisenthal, L. M., Lundeen, D.' E., Bessler, W. and Goldstein, I. J. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1848-1851 3 Reichart, C. F., Pan, P. M., Mathews, K. P. and Goldstein, I. J. (1973) Nat. New Biol. 242, 146147 4 Patterson, R., Suszko, I. M. and Mclntire, F. C. (1973) J. Immunol. 110, 1402-1412 5 Ryser, H. J.-P. (1967) Nature 215, 934-936 6 Larsen, B. (1973) in Chemotherapy of Cancer Dissemination and Metastasis (Garattini, S. and Franchi, G., eds.), pp. 235-243, Raven Press, New York 7 Barfod, N. M. and Larsen, B. (1974) Eur. J. Cancer 10, 765-769 8 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 9 Katchalsky, A. (1964) Biophys. J. 4, 9-41 10 Tareyama, H. (1952) J. Polymer Sci. 8, 243-253 11 Panyim, S. and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346 12 Rosenberg, H., Ennor, A. H. and Morrison, J. F. (1956) Biochem. J. 63, 153-159 13 Rodbard, D. and Chrambach, A. (1971) Anal. Biochem. 40, 95-134 14 Seaman, G. V. F. and Heard, D. H. (1960) J. Gen. Physiol. 44, 251-269 15 Stark, G. R. (1970) Adv. Prot. Chem. 24, 280-283 16 Wallach, D. F. H. (1972) in The Dynamic Structure of Cell Membranes (Wallach, D. F. H. and and Fisher, H., eds.), pp. 181-185, Springer Verlag 17 Nevo, A., de Vries, A. and Katchalsky, A. (1955) Biochim. Biophys. Acta 17, 536-547
© Elsevier ScientificPublishing Company, Amsterdam- Printed in The Netherlands BBA 37260 POLYMERIZATION OF PROTAMINE SULPHATE BY CARBODIIMIDE AND INTERACTION OF ISOLATED PROTAMINE POLYMERS WITH H U M A N RED BLOOD CELLS
N. M. BARFOD and B. LARSEN The Institute of Cancer Research, Radiumstationen, DK-8000 Aarhus C (Denmark)
(Received July 22nd, 1975)
SUMMARY A method using a water-soluble carbodiimide to polymerize protamine sulphate is described. The behaviour of polymerized protamine in Sephadex chromatography and in polyacrylamide gel electrophoresis indicates that protamine has been polymerized into aggregates with defined molecular weights. Turbidimetrical titrations of the isolated protamine polymers with dextran sulphate show that the cationic charge density has been conserved after polymerization. The binding characteristics of the protamine polymers to human red blood cells as measured by cell electrophoresis indicate increased affinity with increased molecular weight of the polymer.
INTRODUCTION It is a well-documented fact that the molecular size of proteins and other macromolecules plays an important role in their biological interactions. Evidence has accumulated of the importance of this molecular parameter in the interaction of cell surface-binding macromolecules such as lectins, antigens and polycations [1, 2]. Thus, cross-linking of soybean agglutinin into high molecular weight complexes with glutaraldehyde caused a 100-200-fold higher specific haemagglutinating activity than that of the native lectin when tested with human erythrocytes [1]. Maximum stimulation of [3H]thymidine incorporation into mouse spleen lymphocytes by cross-linked soybean agglutinin was obtained at a concentration that was five times lower than that required for maximum stimulation by the unmodified lectin [1]. Furthermore, concerning the mitogenic activity of two haemagglutinating compounds (a dimer and a tetramer) purified from the Lima bean (Phaseolus lunatus), the tetramer was several times more active than the dimer form [2]. Reichart et al. [3] found that acetylated concanavalin A, a dimer, possessed about one half the potency of tetrameric concanavalin A as a mitogen for human lymphocytes. The antibody response in guinea pigs and rabbits demonstrated that glutaraldehyde-polymerized ragweed-pollen antigen E exhibited greater antigenicity than the monomeric ragweed antigen E [4]. Finally, the polycation-enhanced uptake of albumin into tumour cells showed increased effect with increasing molecular weight (Mr) of polycations [5], and
198 in parallel with this it was found that the tumour-toxic effect was increased by increasing molecular weight of the polycation [6, 7]. In the elucidation of the significance of molecular size of proteins for interaction chemical polymerization may be employed. Generally, polymerization of proteins is difficult to control since proteins are rather complex molecules with many reactive side groups capable of a multitude of reactions. Recently, we showed that the low molecular weight basic protein protamine from Salmo irideus (iridine) could be polymerized by a water-soluble carbodiimide into defined aggregates, which were separated by Sephadex chromatography [7]. The tumour-toxic effect on JB-1 ascites tumour cells of the partially separated protamine polymers showed increased effect with increasing molecular weight of the aggregates. Finally, it was shown that the increased tumour-toxic effect of the high molecular weight polymers could be correlated with increased affinity for the tumour cell surface as measured by cell electrophoresis. Here we describe an improved method for polymerization of the polycation protamine (iridine) with carbodiimide giving rise to dimers, tetramers, hexamers, etc. Fractionation of the polymers and some preliminary chemical characterizations are also reported. Since polycations bind to the anionic sites on the cell surface, the purified aggregates were tested by their affinity for human red blood cell surfaces as measured by cell electrophoresis. MATERIALS AND METHODS
Polymerization procedure. Protamine sulphate (Nordisk Insulin Laboratorium), 1.0 g, was dissolved in 30 ml of glass-distilled water and cooled to 0 °C. A water-soluble carbodiimide, 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide, (Sigma Chemical Co.), 10.0 g, in 20 ml of glass-distilled water (0 °C) was slowly added. The solution was adjusted to pH 5.0 with 1.0 M HC1. The mixture was allowed to react for 6-10 days in a refrigerator (4 °C). Thin-layer gel filtration. Thin-layer gel filtration was performed with the Pharmacia thin-layer gel filtration apparatus on Sephadex G-75 superfine. The gel layer was allowed to equilibrate for 18-20 h before each run. After completion of the run the sample substances in the gel were transferred to Whatman 3MM filter paper stained with 0.1 ~ Bromophenol blue in water/acetic acid (95:5, v/v) saturated with HgC12 and rinsed in water/acetic acid (95:5, v/v). Molecular weight estimations. Molecular weights of protamine and polymers of protamine were determined by thin-layer gel filtration by developing them together with reference proteins (Mr in parentheses): cytochrome c (12400), myoglobin (17 800), chymotrypsinogen A (25 000), ovalbumin (45 000), bovine serum albumin monomer (67 000), bovine serum albumin dimer (134 000) (Schwarz/Mann, Orangeburg, N.Y.). Reference lines were obtained by plotting the inverse migration distances relative to Blue dextran (2 000 000) against the logarithms of the molecular weights. Column chromatography. The reaction mixture was fractionated on a column (5.0 × 90 cm) packed with Sephadex G-75 superfine. Fractions were monitored by the method of Lowry et al. [8] at 750 nm. All gel filtration procedures including thinlayer gel filtration were performed with 0.15 M NaC1 as eluent. Pooled fractions were dialysed against distilled cold water (4 °C) and lyophilized.
199
Turbidimetric titrations. The charge density of protamine and protamine polymers was compared by titration with the polyanion dextran sulphate (containing 17 :k 0.5 ~o S and 6.5 ~ water). Generally, cationic and anionic polymers react stoichiometrically in diluted solutions [9, 10]. The titration is followed by an increase in turbidity caused by the formation of colloidal polycation-polyanion complexes until the electrochemical point of equivalence is reached. Addition of more polyanion causes a decrease in turbidity owing to precipitation of insoluble complexes. Turbidity was measured at 340 nm after 20 min. Gel electrophoresis. Polyacrylamide gel electrophoresis was performed by the method of Panyim and Chalkley [11]. The gels were stained overnight in 0.1 ~o Coomassie brilliant blue dissolved in 7 ~ acetic acid and destained by diffusion against 7 ~o acetic acid. Cell electrophoresis. Preparation of normal human erythrocyte suspensions and measurements of cellular electrophoretic mobilities were performed as recently described [7]. RESULTS
Chromatography The degree of polymerization resulting from different conditions for the reaction was determined by thin-layer gel filtration in order to trace the optimal circumstances for preparative purposes of polymerized protamine. It was found that for optimal polymerization the reaction requires (l) low temperature, (2) pH 4-5, (3) high concentrations of protamine and carbodiimide, and (4) a reaction time of at least 3 days. Stirring was avoided because this ruined polymerization. Preparative fractionation on Sephadex G-75 superfine yielded three peaks as seen in Fig. 1. The reason why the eluate was investigated by the Lowry method of protein analysis was that protamine as well as carbodiimide were stained by this procedure. The large slow eluting peak is carbodiimide. After dialysing and lyophilizing
2.0
1.5
1.0
! 0,5
I 0 40
60
80
100
120
Fraction number
Fig. 1. Column fractionation of carbodiimide-polymerized protamine on Sephadex G-75 superfine.
200 of the three pooled polymer peaks the fractions were rechromatographed on thinlayer gel filtration. It was found by thin-layer gel filtration that only partial isolation of the polymer classes was achieved. Rechromatography of the highest molecular weight fractions was performed on G-100 superfine Sephadex, and of the more low molecular weight fractions on G-50 superfine. Thin-layer gel filtration of the isolated
!
!
! 0
i,
Startla~ Im Fig. 2. Thin-layer gel filtration of isolated protamine-polymerspecies from carbodiimide polymerization.
fractions yielded nearly complete separation of protamines with homogeneous molecular weights (Fig. 2). Molecular weight estimations based on thin-layer gel filtration showed uniform protamines with approximate molecular weights of 6000, 11 500, 24 500 and 39 000. Judging from these data, we presume that protamine of molecular weight 6000 is polymerized quantitatively into dimers (Mr 11 500), which may condense into tetramers and hexamers. Since we have only six standards there is considerable uncertainty in estimation of molecular weights of polymerized protamine so that these values must be taken as rough approximations.
201 Titrations
Accurate protein analysis of protamine and the isolated polymerizates had to be performed with the Biuret reagent. The Lowry method was not employed because (1) it lacked linearity of standard curves and (2) it stained carbodiimide and might thus interfere with the protein analysis. Carbodiimide did not stain with the Biuret reagent. Amounts of 3.0 ml of 0.1 mg/ml (determined from a protamine Biuret standard curve) of protamine monomer, dimer, tetramer and hexamer were turbidimetrically titrated with 1.0 mg/ml dextran sulphate. The result of the titrations appears from Fig. 3. The almost identical point of equivalence of the titration curves indicates that none of the cationic groups of protamine was lost during the polymerization process. Thus, the only variable molecular parameter of the protamines was the molecular weight. It is seen from Fig. 3 that the titration curve for the higher molecular weight species ascends more rapidly before maximal turbidity, and descends more rapidly beyond the maximum. This may be caused by a higher affinity of the high J
0,60
i
0.50 f
0A0 I i E ¢
0.30,-
0.20 i
di-PS
I i
tetra-PS
0,10 /
hexa-PS
0
50
100
150
200
250
300
ILl 1.0mg/ml Dextran Sulphate
Fig. 3. Turbidimetrical titration of protamine sulphate (PS) and isolated protamine polymers (diPS, tetra-PS, etc.) with dextran sulphate.
202
Fig. 4. Polyacrylamidegel electrophoresis at pH 3.2 in 8 M urea. Right gzl, protarnine; left gel, carbodiimide-polymerizedprotamine.
molecular weight material for dextran sulphate: When dextran sulphate is added to the protamine solution, the high molecular weight species react more readily with dextran sulphate giving more turbidity. Beyond the point of equivalence, the dextran sulphate-protamine polymer complexes precipitate more rapidly because of larger dimensions of the polyanion-polycation complexes. Analysis of the amount of arginine [12], the only amino acid of protamine containing a charged group, yielded 83 g arginine per 100 g protamine base, which gives 28.2 mol of arginine per mol of protamine base (assuming a molecular weight of 6000 of protamine base). Calculations concerning the titration of protamine with dextran sulphate revealed that negatively charged sulphate-esterified groups in dextran sulphate react nearly stoichiometrically with the positively charged arginyl residues in protamine (1.00 #mol arginyl residues were titrated with 1.08/~mol of S), and that no cationic groups were lost during polymerization.
Gel electrophoresis Polyacrylamide gel electrophoresis at pH 3.2 under denaturing conditions in 8 M urea showed that the aggregates were covalently coupled (Fig. 4). From the bands in the upper part of the gel obtained after electrophoresis it is seen that the reaction goes on to produce polymer aggregates with molecular weights beyond the
203
1.0
0.5
0.4
'~ 0.3
0.2
0.1
6 000
12 CO0
24 000
36 000
48 000
60 000
Molecular weightsoipolymerized protamine
Fig. 5. Ferguson plot of mobility versus the expected molecular weights of polymerized protamine (dimers, tetramers, etc.). protamine hexamer. However, it was not possible to isolate detectable amounts of such higher molecular weight protamine polymers. Since the cationic charge density of the protamine polymers is identical as determined from the titration curves, the retardation coefficient KR may be assumed to be proportional to molecular weight of the different polymers. Thus, a linear relationship should exist between the logarithm of the electrophoretical mobility and molecular weight according to Ferguson [13]. Assuming that monomer protamine (Mr 6000) is polymerized into dimers ( M r 12 000), tetramers ( M r 24 000), hexamers (M~ 36 000), octamers (M~ 48 000), and decamers (Mr 60 000), a plot of molecular weight versus log mobility should represent a straight line. This is in fact the case as seen from Fig. 5, although some deviations are observed for monomer and dimer protamine.
Cell electrophoresis As a consequence of high negative cell surface charge [4], red blood cell surfaces bind a variety of positively charged compounds, including protamine and other polycations. The adsorption of polycation on the cell surface is accompanied by a reduction of the negative surface potentials, and even positive values of the surface potential may be obtained with increased concentrations of polycation [6]. Consequently, cell electrophoresis was a convenient technique for studying the binding of protamine polymers to human red blood cells. The electrophoretical mobility (M v/E, where v ~ velocity of the cell in the electrical field and E = field strength) was plotted against the logarithm of the concentration of added polycation as seen in
204
hexa-PS +0,5
"7 :> '7 0.0
/
~
=. =E
-0.5
1
-1.0
Fig. 6. Effect of protamine and protamine polymers on electrophoretical mobility of human red blood cells. Temperature, 25 °C. Measured in phosphate-buffered saline/Sorbitol buffer (1:4, v/v); ionic strength, 0.035; cell concentration 5.105/ml. S.D. for each measurement is shown in the figure. Mobility determinations are uncertain when approaching zero, which is indicated with brackets. Fig. 6. It is obvious that the binding of protamine to the cells was enhanced with increasing molecular weight of protamine since smaller amounts of high molecular weight protamine were required to neutralize the negative surface potential. A relative expression of the affÉnity of the various polymers for red blood cells may be obtained by comparing the amount of polycation necessary to furnish the cell with a given mobility. Since the surface charge should be identical for cells with equal mobility, it is assumed that the number of positive groups adsorbed per cell must be identical for the various polymers in order to obtain a given mobility. The amount of the various polymers which have to be added to give zero mobility of the cells represents the sum of the concentrations of the cell bound polycation, Cbound, and the unbound, free polycation, Cfre~. At zero surface potential the amount of polycation bound to red blood cell surfaces is less than 0.1 #g/ml for l0 7 cells/ml [17]. The total added amounts, the values of which are found from the intersection of the curves with the abscissa (at zero mobility) in Fig. 6, may for practical purposes replace Cfre~ since the value of Cbou,d is negligible. The association constant, Kas ~
Cbound Crree × (available binding sites)
may be replaced by the "relative affinity", 1/Cfree as the concentration of red blood cells, i.e. the number of available binding sites is kept constant. Taking the relative affinity of monomer protamine for red blood cells as unity
205
Mw
40 000
30 000
20 000
10 000
0
I
I
L
i
1
5
10
20
Relative Affinity
Fig. 7. The relation between molecular weight of protamine and relative affinity for human red blood cells.
and the affinities of the polymers relative to that, a plot of molecular weights of the different protamines against the logarithm of the relative affinities represents a straight line as seen in Fig. 7. This type of relation between molecular weight and association constants by interaction points to cooperativity in the binding reaction. If S is a cooperativity constant and Ka"ss is the association constant of the n polymer, the following relation exists K~,~ = S ("- 1) K~s where Ka~s, is the m o n o m e r association constant. This bears resemblance to the properties of the interaction mechanism depicted in Fig. 7. DISCUSSION Generally, carbodiimides react with carboxyl groups at slightly acidic p H levels giving an acylisourea, an activated intermediate that in turn can either rearrange into an acylurea or react with a nucleophile [15]. In the present study we used protamine (iridine) both as carboxyl donor for carbodiimide and as a nucleophile. F r o m a theoretical point of view it is likely that these conditions should lead to peptide-bond formation between the protamine molecules. Since the amino acid composition of iridine, in addition to arginine, consists of only neutral amino acids such as proline,
206 serine, valine and glycine, the only possible point of action for carbodiimide is the N-terminal amino group and the C-terminal carboxyl group. This would account for end-to-end polymerization giving rise to protamine polymers with molecular weights of n x 6000. As already mentioned we find only dimers, tetramers, hexamers, etc., not trimers, nor pentamers. These molecular weights were estimated by thin-layer gel filtration and the assumption of dimerization, tetramerization, etc. was supported by a Ferguson plot (Fig. 5). An explanation might be that protamine is first converted quantitatively into dimers, which subsequently are able to condense into tetramers, hexamers and octamers. The possible linearity of the protamine polymers as a consequence of end-toend coupling could be proved by theoretical calculations of actual measurements of polymer flexibility and shape in solution for dimer, tetramer and hexamer protamine. However, since physicochemical measurements may be more or less subject to interpretations, a more direct proof would be chemical analysis of the amino acid sequences in peptides obtained from monomer and polymerized protamine after protease (e.g. trypsin) digestion. Experiments are under way to test this lastmentioned approach. Polymerization with carbodiimide was found to be superior to formaldehyde or glutaraldehyde polymerization when dealing with basic proteins such as protamine. We found a decreased charge density of protamine after glutaraldehyde polymerization, but the charge density of carbodiimide-polymerized protamine was unchanged as estimated from the titration curves (Fig. 3), and the binding to red blood cells was greatly increased after polymerization. Ryser [5] demonstrated in 1967 that the biological activity of polycations increases with the molecular weight, which was shown with homologous series of polycations as polylysin and DEAE-dextrans with varying molecular weight. A linear correlation was found between the log molecular weights and log albumin uptake into Sarcoma S 180 II mouse tumour cells. It was claimed that the molecular mechanisms underlying this statement were that an interaction of the polymers with the cell membrane was increased in effectiveness by the number of simultaneous attachments to the membrane [5, 9]. The fact that the molecular weight of protamine polymers is proportional to the logarithm of the relative affinity as seen in Fig. 7 points to the presence of cooperativity in this type of interaction [16]. The cooperativity of interaction is characterized by the influence on the binding constant of one group of the polymer to a binding site by the previously bound group of the polymer; K~"ss= S c " - 1 ) K,st s. The similarity of this relation between molecular size and affinity of the binding of polycations to red blood cell surfaces with the experimental data depicted in Fig. 7 may be suggestive for the assumption that cooperative forces are operating. As a result of the formation of such very strong binding forces conformational alterations of the cell membrane are apt to occur, both in the molecular and the structural dimensions, leading to alterations of cellular activity as previously shown [7]. To recapitulate, we have described a method using a water-soluble carbodiimide to polymerize protamine into aggregates with defined molecular weights. Furthermore, we have found a conservation of the cationic charge density of the isolated protamine polymers, which was confirmed by measuring the relative affinity of protamine polymers for the human red blood cell surface. In this manner we have
207 shown that cooperativity may play an i m p o r t a n t role in the interactions of polycations with cell surfaces. ACKNOWLEDGEMENTS We wish to t h a n k Mrs. Jytte Olsen a n d Miss Pia Ohl for the p r e p a r a t i o n of the figures a n d N o r d i s k I n s u l i n L a b o r a t o r i u m for the supply of p r o t a m i n e sulphate. This work was sponsored by the D a n i s h Cancer Society. REFERENCES 1 Lotan, R., Lis, H., Rosenwasser, A., Novogrodsky, A. and Sharon, N. (1973) Biochem. Biophys. Res. Commun. 55, 1347 2 Ruddon, R. W., Weisenthal, L. M., Lundeen, D.' E., Bessler, W. and Goldstein, I. J. (1974) Proc. Natl. Acad. Sci. U.S. 71, 1848-1851 3 Reichart, C. F., Pan, P. M., Mathews, K. P. and Goldstein, I. J. (1973) Nat. New Biol. 242, 146147 4 Patterson, R., Suszko, I. M. and Mclntire, F. C. (1973) J. Immunol. 110, 1402-1412 5 Ryser, H. J.-P. (1967) Nature 215, 934-936 6 Larsen, B. (1973) in Chemotherapy of Cancer Dissemination and Metastasis (Garattini, S. and Franchi, G., eds.), pp. 235-243, Raven Press, New York 7 Barfod, N. M. and Larsen, B. (1974) Eur. J. Cancer 10, 765-769 8 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 9 Katchalsky, A. (1964) Biophys. J. 4, 9-41 10 Tareyama, H. (1952) J. Polymer Sci. 8, 243-253 11 Panyim, S. and Chalkley, R. (1969) Arch. Biochem. Biophys. 130, 337-346 12 Rosenberg, H., Ennor, A. H. and Morrison, J. F. (1956) Biochem. J. 63, 153-159 13 Rodbard, D. and Chrambach, A. (1971) Anal. Biochem. 40, 95-134 14 Seaman, G. V. F. and Heard, D. H. (1960) J. Gen. Physiol. 44, 251-269 15 Stark, G. R. (1970) Adv. Prot. Chem. 24, 280-283 16 Wallach, D. F. H. (1972) in The Dynamic Structure of Cell Membranes (Wallach, D. F. H. and and Fisher, H., eds.), pp. 181-185, Springer Verlag 17 Nevo, A., de Vries, A. and Katchalsky, A. (1955) Biochim. Biophys. Acta 17, 536-547
