Kinetic regularities of the degradation of collagen in dilute solutions of sulphuric acid and potassium hydroxide

Polym~ Science U.S.S.R. Yol. 22, No. 10, pp. 25832590, 1980 Printed in Poland

0032-3950/80[102585..-06507.5010, © 1981PergamonPre~ Ltd.

KINETIC REGULARITIES OF THE DEGRADATION OF COLLAGEN IN DILUTE SOLUTIONS OF SULPHURIC ACID AND POTASSIUM. HYDROXIDE* L. G. 1)RIVALOVA,M. L. KONSTAI~TII~IOVA,V. N. KuI~GI!~, G. YE. ZAIKOV, Yx. YA. SOROKI~ and G. S. DREIZE~SHTOK I n s t i t u t e of Chemical Physics, IJ.S.S.R. Academy of Sciences Leningrad Branch of the All-Union Scientific Research Institute of Synthetic Fibres

(Received 27 September 1979) A study was made of kinetics of degradation of complex collagen fibres treated with chromium salts, in water and in dilute solutions of sulphuric acid and potassium hydroxide in the p H range of 0-3 and 12-14 at 30-60 °. The diffusion coefficient of water vapour into collagen K ~ l ° = 7 + 0 . 5 × 10-* cm2/sec was determined. I t was shown ~hat the degradation of collagen in acid and alkaline media takes place in the " i n t e r n a l " kinetic region and breakdown of the polymer takes place at random. Effective rate constants were derived for the hydrolysis of collagen a n d it was shown that the effective activation energy of the process is practically independent of the concentration of alkali a n d acid a n d is 20=t=1 kcal/mole.

CXT(~UT thread, the main component of which is collagen, is widely used in medical practice as reabsorbing joining material. However, its mechanism of degradation in the organism has not been studied in practice. It is therefore a d v i s a b l e to e x a m i n e the d e g r a d a t i o n of collagen in a m o d e l m e d i u m , in o r d e r t o e s t a b l i s h m a i n k i n e t i c r e g u l a r i t i e s o f t h e process. T h e s e r e s u l t s a r e n e c e s s a r y for creating samples w i t h a controllable r a t e of resorption. Collagen was used for the study with cattle sinew as raw material. After preliminary t r e a t m e n t in a solution containing 100 g/1. NaOH and 142 g/1. Na~SO~ and neutralization with 3~o boric acid collagen was dissolved in 1 • acetic acid and 2% spinning solution obtained which was spun into acetone containing ammonia (0-07%) and formaldehyde (0"15%). T r e a t m e n t of collagen, in order to increase its resorption time in tile organism, was carried out for 3-5 hr in a 5~o solution of Na~SO4 containing a chromium extract ~vith a basicity of 38% and a concentration of 50/o (in terms of Cry03) at 25°. The samples examined were complex fibres of average diameter 100 /lm. Experiments were carried out in dilute solutions of sulphuric acid and potassi~Lm hydroxide in the p H range of 0-3 a n d 12-14 a n d in water at 30-60 ° . Collagen does not adhere under these conditions a n d the hardener used does not protect it from acid [1] or alkaline [2] hydrolysis. Kinetics of degradation were followed from the weight variation of samples; at t i m e intervals they were removed from the reaction cell, washcd with water and dried in a vacuu m drier to constant weight. * Vysokomol. soyed. A22: No. 10, 2354-2358, 1980. 2585

L. O. Pm~ALOVAet

2586

02.

As an example Fig. 1 shows typical kinetic curves of weight variation of samples during degradation in acid and alkaline media. Adsorption studies show that at 25 ° collagen is rapidly saturated with water. The diffusion coefficient of water vapour calculated from experimental results is 7 + 0.5 ~( 10-9 cm=/sec. This suggests that the process takes place in the " i n t e r n a l " kinetic region [3]. Analysis of results concerning relative rates of decomposition of dipeptides in acid and alkaline media (Table 1) indicates that peptide bonds somewhat differ in reactivity and it may be assumed that the decomposition of collagen--a high molecular weight polypeptide--takes place at random. too- m ~ 100 %

I

/'rl o

•

3

©

3

20 l

I00

200

' 800 10 T i m e ~rain

30

50

Fie. l. Kinetic curves of gravimetric loss of collagen samples in 0.1 ~ solutions o f sulp h u r i e acid (a) and potassium hydroxide (5) at temperatures of: a--58.5 (/), 55 (2), 50 (3) a n d 40 ° (4); b - - 5 0 (1), 40 (2) and 30 ° (3).

I f the probability of bond rupture is independent of its position in the macromolecular chain, with homogeneous hydrolysis of polymers the effective raCe constant k is determined from the equation [6] _lnP--1

P

inP~--I =

-

+kt,

(1)

where P0 and P are the average degree of polymerization of the polymer initially and at time t. The gravimetric proportion y of the polymer formed with x units is calculated from the equation x

where a = l - - e -~t is the probability of bond rupture; 1--a--the probability of retention of the bond. The first term in eqn. (2) allows for polymer decomposition with rupture of one bond and the second t e r m - - t h a t with the simultaneous rupture of two bonds. We assume that the probability of decomposition with the simultaneous rupture

Kinetic regularities of degradation of collagen

2587

of two bonds is slight, compared with the rupture of a single bond and t h a t ( 1 - - a ) ~ - l = l since the weight variation of collagen samples takes place as a T A B L E 1. R E L A T r V ~ RATES OF HYDROLYSIS ~) OF D I P E P T I D E S I N ACID [4] AND A L ~ A L r N E [5] MEDIA (Wglyeyl_glyetne~ 1)*

No.

Dipeptide DL-alanine-glycine Alanine-leucine Glycine-L-alanine Glycine-D-alanine Glycine -leucinc Glycine-tyrosine Leucine-aspartic acid Leucine-glutamic acid

w

No.

0.69 0.32 0.37 0.40 0.34 0.43 0.86 0.23

9 10 11 12 13 14 15

Dipeptide I~ucino-glyeine Serine -alanine Seryl-serine Serine-glycine Glycine-DL-lcucine Glycine-DL-valine Glycine-DL-a-alanine

w 0"23 0.74 0.40 0 "40 0.23 0.14 0"39

* Conditions of hydzolysis: 1-42=2 N HC1, 99°; 13-1510.05 1~ XOH, 88 °.

result of degradation, resulting in the formation of particles with a limiting degree of polymerization x, which change into solution. Then, eqn. (2) takes the form ~x=2a ~ = 2

(1--e-**)

or considering eqn. (1) after transformation we obtain

~,=2 P \ 1-I/Po /

(3)

In eqn. (3) the value of I/Po<>1000 [I]. Therefore,

According to the Kulm formula

Hen

1

1

P

P0

=kt

(4)

• kt

(~)

ce ?x= 2 -P

After substitution into eqn. (5) of the value of P from formula (4) we obtain xkt

~=2xku2+2 p-~

(6)

2588

L.O.

PRrVALOVA et al.

Considering that the second term of eqn. (6) is low, compared with the first, we have ~ = 2xk2t 2 (7) Products with a degree of polymerization of 1 to x transfer from collagen into the solution. Therefore, eqn. (7) finally takes the form (8)

)'x= 2k~tSSx,

where S~ is the total of terms of the arithmetical progression. As shown by Fig. 2, in coordinates ?~--t ~ kinetic curves are linear. Effective rate constants of hydrolysis of collagen kat=x/tan =/2Sx determined from these dependences for various. process conditions are tabulated (Table 2). T~B~

2. E r r z O = V E ~ T ~

COlCSZA~TS OF aYDROLYSZS OF COLI~O~.N ZN A c r e ArCD ,T,waza~r. MEDIA I

Kef r × l 0 s (rain -t) a t g i v e n p H v a l u e s

TO 0 30"0 37-7 40"0 50.0 50.7 55-0 57-0 58-5 60-0

1

I

2

7'4

3

I

12

13

14

2"19

10"7

49.0

0-05 3.03 7.7 14.0 15.7

0.83 2"1 -4.2

0.15 0.41 0.64

--

1"18

0.12 0.36

6"3 16"4

0.08

28"7 76

-j

133 3O0

0.17

According to results [7], the average molecular weight of collagen particles dissolved during hydrolysis shows little dependence on temperature. This enables the effective activation energy of the process to be calculated. Activation energies of collagen degradation are practically independent of the concentration of alkali

7~ 0"5'~

I

o,q-

.

2

0.2

lO

3O

50

70

fz~ lO-Z,~in2

FzG. 2. Graphical solution of o q n . (8): 1 - - 0 . 0 1 ~ K O H , 2--1 ~ H~SO¢; 7x=(mo--m)/mo, 50 °.

Kinetic regularities of degradation of collagen

2589

a n d acid and are 20+ 1 kcal/mole. The average molecular weight of soluble collagen particles in the p H range of 0-3 and 12-14 is 2500-3000 [7]. I f we assume t h a t the average molecular weight of aminoacid residues is 100, the degree of polymerization of soluble collagen particles is 1-30 units. Bearing in mind this value, effect~ive rate constants of collagen hydrolysis show satisfactory agreement with effective rate constants of hydrolysis of peptides [8]. TABLE

3. R A T E

CONSTANTS OF H Y D R O L Y S I S O~ P E P T I D E S

I~ 1 N

Rate constants of hydrolysis k×10 ~, min -1.

T° 94"2 74"6 55"6

IN 2 N ~IC1 AND OF C O L L A G E N

H2SO4

]~g--D

kg-g

kl-g

kg--1

klg--p

3"87 0"72 0"12

6"42 1"46 0"23

1-45 0"27 0"04

3"00 0"61 0-11

15"35 3'11 0"55

kg-gp

]¢l-gl

kg--lg

8"21 1"64 0"27

2"17 0"35 0.05

3"50 0"70 0"13

kc

9"52 2"10 0"46

* g -- glycine, p -- phenylalaniue, 1-- leuciae and c -- collagen.

Table 3 shows rate constants of hydrolysis of some di- and tri-peptides at different temperatures. This Table shows, for comparison, rate constants of hydrolysis of collagen, calculated by us for the same temperatures. I t can be seen t h a t these are values of the same order of magnitude. I n dilute solutions of acids and bases the variation of the effective rate constant of hydrolysis of amides, according to catalyst concentration, is described by the equation ~eff~-~true"

¢cat

where ktrue is the rate constant of hydrolysis independent of the composition of the catalyst medium; Ccat--concentration of solvated H+ and O H - ions. According to this equation there is a linear relation between log ke~ and medium pH: the t a n g e n t of the gradient of this straight line is equal to unity. However, for collagen hydrolysis the linearity of the dependence is retained, but the tangent of the gradient for hydrolysis in acid medium is 0.5 and in alkaline medium, 0.7. The difference of these values from one is, apparently, due to the fact t h a t in the polymer matrix catalyst concentration is different from t h a t in solution, as indicated by Tanford [9]. Translated by E. S ~ . ~

REFERENCES

1. A. N. I~]KHAILOV, Kollagen kozhnogo pokrova i osnovy ego pererabotki (Collagen of the Skin and Principles of Treatment). Izd. "Legkaya industriya", 1971 2. N. R A T Z E R a n d H. GR~NEWALD, Makromolek. Chem. 9: 116, 1953 3. Yu. V. MOISEYEV, V. S. MARKIN and G. Ye. ZAIKOV, Uspekhi kbimii 45: 510, 1976 4. R . I~H.L, Adv~mcesProtein Chemistry 20: 37, 1965

2590

A.V.

OLENI~ d aZ.

5. I. G. ORLOV, K a u d i d a t s k a y a dissertatsiya (Post-Graduate Thesis). Moscow, I K h F , U.S.S.R. Academy of Sciences, 1966 6. N. M. EMANUEL' and D. G. KNORRE, Kurs khimicheskoi kinetiki (Course on Chemical Kinetics), Izd. "Vysshaya shkola", 1974 7. G. CHIRITA and A. CIOBANU, Ind. usoara 18: 605, 1971 8. D. A. LONG and T. G. TRUSCOTT, Trans. F a r a d a y Soc. 59: 918, 1833, 2316, 1963; D. A. LONG a n d J. E. LHJLYCROP, Trans. F a r a d a y Soc. 59: 907, 1963 9. I. TANFORD, Fizicheskaya khimiy~ polimerov (Physical Chemistry of Polymers). Izd. " K h i m i y a " , 1965

Polymer Science U.S.S.R. %'oi. 22, ~o. 10, pp. 2590-2597, 1980 Printed in Poland

0032-3950/80/102590-08507.50/0 © 1981 PergamonPrvmLtd.

SYNTHESIS OF GRAFT POLYMERS AND BLOCK-COPOLYMERS OF ACRYLIC AND METHACRYLIC MONOMERS BY A "LIVING" RADICAL POLYMERIZATION* A . V. 0LENII~, A . ~B. KHAINSON, V. ]~. GOLUBEV, l~I. B. L A c H r N o v , V. P . ZV-BOV a n d V. A . KABANOV M. V. Lomonosov State University, Mosco~v

(Received 27 September 1979) I t was shown t h a t in the presence of a complex-forming agent--o-phosphoric acid radical graft polymerization of methyl metahacrylate or methyl-a-chloraerylate on a F-irradiated cellophane film takes place b y a mechanism of "living" c h a i n s . A t the initial stage of the reaction with a practically constant number of graft chains under given conditions graft polymer yield and the molecular weight of graft chains increases in direct proportion to the time of contact of a y-i,wadiated film with the reaction system. A s t u d y was made of the dependence of graft polymer yield, molecular weight and the number of PMMA graft chains, on the composition of t h e H,PO4-MMA system. The r a t e constant of chain propagation was evaluated when carrying out graft post-polymerization of MMA on a r-irradiated cellophane film. I t was shown t h a t the existence of " l i v e " chains in the grafting of methyl methacrylate and methyl-a-ehloraerylate in the presence of HsP04 on to y-irradiated cellophane film enables graft block-copolymers and graft tri-bloek copolymers of these monomers to be obtained with control of the molecular composition and the length of each unit. The block structure of individual graft chains was confirmed by turbidimetrie titration. Block-copolymers of methyl methacrylate and methyl-~-chloraerylate practically free from homopolymers were obtained b y hydrolysis of this modified cellophane film. * Vysokomol. soyed. A22: :No. 10, 2359-2365, 1980.