Study of the polycondensation of biopolymer macromolecules

N. P. KUZNETSOVAand G. V. SAMSONOV

2934

8. N. A. K H O K H R Y A K O V A , B. G. BAL'KOV, N. I. M I T S K E V I C H , N. G. ARIKO and V. I. KO-

VALEV, U.S.S.R. Pat. 8830006, Byul. izobret., 43, 62, 1981 9. N. R. P R O K O P C H U K , Izv. AN BulgSSSR, seriya fiz.-tekhn.-nauk, 4, 62, 1981

10. N. R. PROKOPCHUK, Plast. Massy, 10, 24, 1983 11. S. A. BARANOVA, P. M. PAKHOMOV, B. N. KLYUSHNIK, S. A. GRIBANOV, V. E. GELLER and M. V. SHABLYGIN, Vysokomol. soyed. B23: 104, 1981 (Not translated in Polymer Sci. U.S.S.R.) 12. A. VLOKHOVICH and M. LINEK, Acta Polymerica 32: 445, 1981 13. G. M. BARTENEV, S. N. KARIMOV and D. SEMATOV, Acta Polymerica 34: 44, 1983 14. M. PRASADARAO and E. J. PEARCE, Polymer Sci. Polymer Chem. Ed. 20: 1669, 1982 15. Kratkii spravochnik fiziko-khimicheskikh velichin (Shorter Handbook of Physico-Chemical Quantities) p. 200, edited by K. P. Mishchenko and A. A. Ravdel', Khimiya, Leningrad, 1972 16. J. PIMENTELL and O. MaeCLELLAN, The Hydrogen Bond, p. 462, Mir, Moscow, 1964 17. V. L VETTEGREN, A. A. KUSOV, L. N. KORZHAVIN and S. Ya. FRENKEL', Vysokomol. soyed. A24" 1958, 1982 (Translated in Polymer Sci. U.S.S.R. 24: 9, 2241, 1982) 18. T. G. KYAZIMOVA, M. M. GUSEINOV, R. S. BABAYEV and E. G. ASLANOVA, Plast. Massy, 3, 53, 1983

Polymer Science U.S.S.R. Vol. 27, No. 12, pp. 2934-2939, 1985

Printed in Poland

STUDY

0032-3950/85 $10.00+.00 © Pergamon Journals Ltd.

OF THE POLYCONDENSATION OF BIOPOLYMER MACROMOLECULES* N. P. KUZNET$OVA a n d G. V. SAMSONOV High Polymer Institute, U.S.S.R. Academy of Sciences (Received 7 May 1984)

The polycondensation of a protein with a bifunctional crosslinking agent has been investigated, taking as an example'the interaction of a serum albumin with glutaric aldehyde. By varying the concentration of the initial components it is possible to direct the process towards intra- or intermolecular crosslinking of the albumin macromolecules. The apparent MWD of the oligomer products of polycondensation (oligoalbumins) has been investigated in relation to the polycondensation conditions. To calculate molecular weights with different degrees of averaging (_~rw and 3~tn)it is proposed that a method of gel permeation chromatography for oligomeric protein macromolecules should be used. DURING the last decade processes of p o l y c o n d e n s a t i o n in systems o f the type p r o t e i n b i f u n c t i o n a l crosslinking agent have frequently led to study o f m a n y biological phen o m e n a [1-4]. As one of the c o m p o n e n t s we have a p o l y f u n c t i o n a l b i o p o l y m e r (protein) which, despite the monodispersity a n d c o n f o r m a t i o n a l u n i f o r m i t y of its macromolecules *Vysokomol. soyed. A27: No. 12, 2611-2614, 1985.

Polycondensation of biopolymer macromolecules

2935

leads to significant complication of the polycondensation process. When interacting with albumin macromolecules bifunctionai reagents are capable of forming intramolecular chemical bridges between protein functional groups that are located sterically close to one another. Reactive functional groups may also form parts of various protein macromolecules, with the result that oligomers may be formed at the expense of intermolecular covalent bonds of a single type of protein, or heteromers may be formed if proteins of different types are participating in the reaction [5]. Glutaric aldehyde (GA) is a bifunctional reagent that is in very good supply [6]. In weakly acid aqueous solutions G A exists in a state of equilibrium with the hydrated linear tbrms and the cyclic ones, while in neutral and weakly alkaline solutions G A itself undergoes aldol condensation and subsequent dehydration, resulting in ~,//-unsaturated aldehydes. The chemical inhomogeneity of G A complicates its reaction with proteins. The mechanism of polycondensation of proteins with G A is not known at present, though it has been established that aldehyde groups react preferentially with e-aminogroups of lysine residues of the protein. Several authors have shown that modification of amino-groups of protein macromolecule takes place with the formation of a conjugated Schiff's base, where the aldime bond C H = N formed as a result of the protein NH2 group interacting with the aldehyde group is conjugated with the C = C double bond, making the reaction product stable towards hydrolysis [1, 7]. During polycondensation the functional groups all react independently, so oligomers are normally obtained as a result of the reaction [8]. At the same time two types of structure may be formed both at the expense of intramolecular crosslinking, and on account of the formation of intramolecular bonds resulting in water-soluble high-molecular weight products. The formation of intra- and intermolecular crosslinks depends on the total number of reactive amino acid residues, and especially on their distribution on the surface of the protein globule. If the surface distribution is uniform the intermolecular reaction takes place quite readily, but in the case of a localized distribution an intramolecular iriteraction will tlaen be preferential [9]. In the present investigation we took a serum albumin as an example in an attempt to investigate the polycondensation products of a protein with GA. The serum albumin was that of human placenta blood (a 10~ sterile solution). Albumin lends itself readily to modification as it contains a large number of lysine residues (55) based on the amino acid composition (the MW is 69 x 103) [10]. It dissolves readily in a wide range of concentration and pH. Serum albumin used in the investigation underwent a painstaking dialysis. Investigations took place in phosphate buffer solutions (pH 5-7-7-9), where the native globular conformation of the albumin was preserved. Determination of the number of amino groups in the albumin was based on the reaction with 2,4,6-trinitrobenzenesulpho acid [11]. The amount of modified amino groups was estimated from the difference between the number of amino groups determined in the initial and the modified albumins. Glutaric aldehyde was prepared by vacuum distillation of a commercial 25 ~ aqueous so,ut~on of GA (Reanal grade). An estimate of the amount of aldehyde groups was obtained using the methoa of differential pH-metry with hydroxylamine [12], assuming that the GA molecule (M=100) contains two aldehyde groups. The amount of bifunctional crosslinking agent linked to the albumin

2936

N . P . KUZNETSOVAand G. V. SAMSONOV

was calculated as the difference between concentrations of the initial GA and the amount that had failed to react within a fixed period of time. To do so rapid separation (under slight pressure) of the solution was carried out in the ultracentrifuge cell, using a UM-10 membrane (Diaflow) through which free GA passed readily, while the modified albumin was completely blocked. An accurate test of MW and of the number of albumin NH2 groups was carried out by stopping the polycondensation reaction by adding sodium boron hydride. The polycondensation of the albumins with GA results in a large number of macromolecules that are polydisperse in respect to size and heterogeneous as to their form. A mixture of albumin oligomers may be regarded as a polymers homologous system, and as in the case of any polydisperse polymer systems one may use various methods of molecular weight averaging, i.e. one may use the number-average ~¢, and the weight-average AT/wmolecular weights. To obtain a relative estimate of the MWD of the albumin oligomers we used a GPC method~ to determine )l~r,~and 2~', namely the one that has been proposed for determination of the apparent MWD in a multicomponent low molecular system (a mixture of peptides of a protein hydrolysate) [131. To investigate the systems we used as gel phase Sepharose 6B with a separative power in respect to proteins in the range M = 2 5 x 103-106 equilibrated with a 0.033 M phosphate buffer solution (pH 7"1). Figure 1 shows gel chromatograms of the size distribution of macromolecules of the oligoalbumin solutions. Elution curves for the oligoalbumin were tested by spectrophotometry. The calculating procedure for .,~rw and 217/, envisages prior column calibration according to monodisperse albumin markers of known MW* whose coefficients of distribution Kov on Sepharose 6B had to be determined from the normal ratio of Kay= (lit- Vo)/(llt- Vo),where Vo is the retention volume for the column (using dextran blue); V, is the pore size for the gel (determined from KI); Vt is the elution volume for the marker or for any i component with M=Ma. Parameters Vo and Vt are constant for the column in question. Constants R and S were determined using a calibration curve plotted on the basis of the empirical equation - l o g Ka~=RM2/3+S [13]. It was assumed in the calculations that the size distribution is Gaussian in character. In a mixture of polymer-homologues the concentration of oligomers of any type M=Mt was assumed to be proportional to the optical density D~. Using a gel chromatogram on the coordinates Dr-V~ transformed into a Dz-M~ plot in line with the formula

Vt- VO_s)312 - log ------~"o

Mr=

k'~R

one obtains the MWD pattern for an oligoalbumin. On the basis of an MWD plot for a mixture of albumin oligomers we may determine the .~rw and 3~, values, starting with the following relations:

= Z D,/Z W,/ M,), m - Z o,M,/Z o,. For polydisperse samples ()~rw> ~ r ) the M~,/M. ratio characterizes the degree of polydispersity, At the end of the polycondensation reaction the average MW ratio is usually expressed as )~r ].~r~ = 1 : 2 [8]. The intensity of the crosslinking process, the size of the resulting macromolecules. a n d the degree of a l b u m i n c o n v e r s i o n to oligomers were evaluated first of all in r e l a t i o n to the c o m p o n e n t s c o n c e n t r a t i o n ( a l b u m i n a n d GA), their ratio, a n d the p H value. T h e degree o f a l b u m i n conversion to oligomers ~ d u r i n g p o l y c o n d e n s a t i o n was de-

* The authors thank T. M. Taratin for kindly plotting the calibration curve.

Polycondensation of biopolymer maeromolecules TABLE 1.

2937

POLYCONDENSATION CONDITIONS AND CHARACTERISTICS OF THE RESULTING OLIGOALBUMINS-

(OA) CA × × 1 0 3, m

COAx 102, m

Molar ratio GA:A

0.5 0.9 1.1 1.8 3.0 4-2 6-1

13"5 23'8 29"7 48'6 83"3 82"3 82"0

0'37 0"37 0"37 0"36 0"35 0'51 0'74

o~-~-

[OAI [AI

0.60 0.65 0-73 0-74 0.84 0.87 0.93

GAbouna, % on original 100 93 77 70 70 55 48

Molar ratio Gbou.d:A 8"2 10-6

~ w × l O -~ aT/. x 10 -3

Mw/M~

205 237

106 115

1.93 2.06

259 28l 296 335

123 133 153 170

2.10 2.11 1.93 1.97

I

11"0 20"8 14-9 9-0

t e r m i n e d as the a m o u n t of high molecular weight a l b u m i n derivatives relative to the initial m o n o m e r . T a b l e 1 gives the quantitative results in regard to p o l y c o n d e n s a t i o n o f a l b u m i n with G A (for Fig. 1). A sixfold increase in the initial a l b u m i n / G A m o l a r ratio (from 13-5 to 83) in the reaction system intensifies the process of modification of the

D 0"3 -

2

II I I I

0.1

b

/,~~ 0-3

0.3 I I

30C

20O

\

07

I

2

lOOi 80

V~ml

0-#

03 c A , 10, hi

vo FIG. l

FIG. 2

FIG. 1. Size distribution of oligometric albumins during gel chromatography on Sepharose 6B, varying the albumin concentration (molar ratio GA : A = 82) (a) and varying the ratio of the components ([A] = 0.37 x 10- a m) (b): 1 - initial abuminn; 2 - 7 - oligoalbuminsprepared with [A] = 0"74 x x 10 -a (2); 0.36x 10-am (3); molar ratio GA: A=13"5 (4); 29"7 (5); 48.6 (6); 83-3 (7). FIG. 2. Influence of albumin concentration on MW of albumin oligomers (CGA=0"38 m): !-- Ar/'w, 2--~,.

:2938

N. P. KUZNETSOVAand G. V. SAMSONOV

albumin: the ratio of the amount of G A bound to albumin to the amount of albumin increases from 8.2 to 20.8 which is accompanied by an insignificant increase in ~ M W of the oligomers. When the albumin concentrations are relatively low (0.37 x 10-3 m) G A is expended mainly in intramolecular modification of the albumin, and tri- and tetramers (~tw=205× 103-280x103) are formed at the expense of intermolecular bridges. An increase in the albumin concentration from 0.36 × 10 -3 to 0.74x 10 - s m intensifies the process of oligomer formation while simultaneously reducing the relative amount of bound bifunctional crosslinking agent from 20.8 to 9. The influence of the initial albumin concentration on the M W of the oligomers is illustrated in Fig. 2. TABLE 2. MODIi:ICATIONOF AMINOGROUPSOF ALBUMINDURINGPOLYCONDENSATIONOF GA AT 10° E Modified NH2 groups : albumin

× 1"12 • 1"7 1'12 3"1

2~wX 10-3

3"7 11-0

184 175

0.62 0.62

×

Modified NH2 groups : albumin

,~/w x 10- a

2.7 5.5

5'6 15.2

138 141

TABLE 3. INFLUENCEOF pH ON THE ~POLYCONDENSATIONOF ALBUMINWITH GA (CA=I'12X 10 -3 m, 10°) pH

caA x 102, m

5"7 6"5 7"9 6-0 7-6

3"1 3"1 3.1 1"7 1.7

Modified NH2 groups : albumin 9.6 11-0 11.5 3.7 4.0

Original GA/albumin ratio 27.7 27.7 27.7 15.2 15.2

.h~tw× 10 -a

_~n × 10-3

Mw/Mn

201 175 157 184 169

106 98 75 103 97

1.90 1.78

2.10 1-79 1.74

This means that an increase in the albumin concentration leads to a higher yield of oligomer components, while an increase in the G A concentration leads to intensification of intramolecular crosslinking. The data in Table 2 relate to modification of albumin amino groups accompanying variation in the polycondensation conditions. Where the albumin concentration is constant a doubling of the amount of bifunctional crosslinking agent leads to a threefold increase in the degree of modification o f aminogroups, but has scarcely any influence on MW. In the studied p H interval (5.7-8.0) in which the native albumin conformation is preserved the p H value has only a rather weak influence on the number of amino-groups modified by GA, and likewise on the MW (Table 3). As the p H rises the number of modified amino-groups increases, and at the same time intramolecular modification is more preferential than intermolecular modification (there is even a reduction in the average MW). We surmise that when the albumin becomes further removed from the i s o -

Thermal degradation of elastomers modified with sulphenyl chlorides

2939

electric point (pH 5.0) albumin globules are so to speak "pushed apart" on account of the higher total negative charge on the protein molecule, an effect favourable mainly to intramolecular modification. Translated by R. J. A. HENDRY REFERENCES 1. K. P E T E R S a n d R. M. RICHARD, Ann. Rev. Biochem. 46: 523, 1977 2. R. H E R M A N N , R. JAEKICKE a n d R. R U D O L P H , Biochemistry 20: 523, 1981 3. F. P I T T N E R , T. MIRON, G. P I T T N E R a n d M. J. WILCHEK, Solid-Phase Biochem. 5: 147, 1980 4. M. M. S H T I L ' M A N , Uspekhi khimii 48: 2061, 1979 5. J. N. B A R B O R I N a n d B. T H O M A S S E T , Biochim. Bophys. Acta (E) 570:1 l, 1979 6. D. H O P W O O D , (book) Fixation in Histochemistry (Edited by P. J. Stoward), p. 47, C h a p m a n and Hall, London, 1973 7. P. MONSAN, G. P U Z O a n d H. M A Z A R G U I L , Biochemie 57: 1281, 1975 8. S. Ye. BRESLER and B. L. YERUSALIMSKII, Fizika i khimiya makromolekul, N a u k a , Moscow-Leningrad, 1965 9. J. PAYNE, Biochem. J. 135: 867, 1973 10. G. N E U R A T and K. BAILEY, Belki (Albumins), vol. 3, p. 324, Soviet Foreign Lit. Press, 1958 l l . R. FIELDS, Biochem. J. 124: 581, 1971 12. H. R. ROE a n d G. M I T C H E L , Analyt. Chem. 23: 1758, 1951 13. N. C A T S I M P O O L A S , Analyt. Biochem. 61: 101, 1974

Polymer Science U.S.S.R. Vol. 27, No. 12, pp. 2939-2945, 1985 Printed in Poland

STUDY

0032-3950/85 $10.00+.00 © Pergamon Journals Ltd.

OF THE THERMAL DEGRADATION OF ELASTOMERS MODIFIED WITH SULPHENYL CHLORIDES*

Z. G. SHAMAYEVA,YU. B. MONAKOV,A. M. SHAKIROVAand G. A. TOLSTIKOV Chemistry Institute of the Bashkir Affiliated Branch of the U.S.S.R. Academy of Sciences

(Received 7 May 1984) The behaviour of modified elastomers containing various functional groups has been investigated under conditions of high temperature degradation in an inert medium and in air. It was found that dissimilarities in thermal stability observed for the modified polymers are due to the nature of the elastomer matrices, and to the influence of functional groups in polymer backbones. It has been established that sulphenyl chlorides (in amounts of up to 5 mole %) have a stabilizing influence on thermal degradation of the elastomers. *Vysokomol. soyed. A27: No. 12, 2615-2619, 1985.