Polycondensation of amino acid phosphoanhydrides

14 BBA

BIOCHIMICA ET BIOPHYSICA ACTA

35061

POLYCONDENSATION OF AMINO ACID P H O S P H O A N H Y D R I D E S . II. P O L Y M E R I Z A T I O N OF P R O L I N E A D E N Y L A T E AT CONSTANT P H O S P H O A N H Y D R I D E CONCENTRATION M. P A E C H T - H O R O W I T Z AND A. K A T C H A L S K Y

Weizmann Institute of Science, Rehovoth (Israel) (Received N o v e m b e r 8th, 1966)

SUMMARY

This study is devoted to the polymerization of proline adenylate to oligopeptides. The polymerization was carried out at p H 8.5 maintaining a steady-state concentration of the active phosphate. It was found that every degree of polymerization reaches a steady concentration after a relaxation time increasing with the degree of polymerization. From the time dependence of the concentrations and the steady level it was possible to evaluate the hydrolytic constant k~ = o.I min -1 and the polymerization constant for dimer formation k 1 ~ 3.65 1-mole-1- min -1, for trimer formation k 2 = 5.4 l'mole-l"min-1, for tetramer k8 = 8.8 1- mole -1.rain -~ and for the pentamer k 4 ~ Io. 7 1.mole-~.min -~. For higher degrees of polymerization the constants approach an approximately equal k value. Alternative runs of polymerization with different yields could be obtained by adding free proline to the solution of phosphoanhydride. All the results follow the same theoretical description and can be adequately represented b y the same set of rate constants.

INTRODUCTION

Adenylates of amino acids are well-known precursors of the compounds with t R N A which participate in protein synthesis. The adenylates are labile substances which liberate about io kcal/mole upon hydrolysis. Since the formation of a peptide bond requires approx. 3 kcal/mole, polycondensation m a y proceed spontaneously under physiological conditions in aqueous solution, at room temperature and at p H values close to 7. Since the polycondensation proceeds through the interaction of phosphoanhydride groups with non-ionized amino groups, appreciable polycondensation takes place only at p H ' s higher than 7. In the acid range the adenylates hydrolyze without peptide formation. The polymerization of proline adenylate was studied for the interest in polyprolines related to collagen. As pointed out in the previous paper of this series, it is advantageous to carry out the polymerization in such a way that the concentration of active phosphate remains constant throughout the process. This polycondensation is amenable to a simpler kinetic analysis and can be readily checked against theoretical Biochim. Biophys..4cta, 14o (1967) 14-23

POLYMERIZATION OF PROLINE ADENYLATE

15

prediction. The aim of the present paper is to determine the kinetics and to evaluate the rate constants for different degrees of proline polymerization. EXPERIMENTAL

Analytical methods Determination of the anhydrous bond. I t was found that the most suitable method for our purpose is the hydroxylamine test as adapted b y LIPMANN ANn TUTTLE1 and modified b y HESTRIN2. The determination according to HESTRIN measures both the contents of anhydride and of ester groups. Since some migration of the amino acid m a y take place from the phosphate groups to the hydroxylic groups of the ribose residue, a certain fraction of proline adenylate m a y be in the form of an ester and could not be distinguished by the hydroxylamine test from the phosphoanhydride. The rate of hydrolysis of the ester groups is, however, so much lower than that of the anhydride that there is no difficulty in distinguishing between the two b y kinetic methods. Thus, in the following we base our anhydride determination on HESTRIN'S method, corrected for the ester group as determined b y rates of hydrolysis. Total j~hosj~horus was determined b y the method of SUM~ER3.

Proline and J~roline pe~tides : The determination of proline contents was carried out colorimetrically b y the ninhydrin method. Since proline gives a yellow color, whose intensity can be determined only with difficulty, we used the method of TROLL AND LINDSLEY4 as modified b y ROGOZINSKY*.According to this method a pink color is developed in acid medium whose intensity is directly proportional to the proline concentration. Chromatographic separation of the proline peptides was carried out on Whatman No. I paper with a n-butanol-acetic a c i d - w a t e r (5:1:4, b y vol., upper phase) mixture as solvent. When the moving front reached the bottom of the paper the chromatogram was dried and dipped into a solution of 75 mg ninhydrin in a mixture of 15 ml acetic a c i d - I ml H3PO 4 and 15 ml of n-butanol. The paper was then air-dried and baked in an oven at 85 ° for 20 min to let the spots develop. The RE values for proline and its peptides under the present conditions are the following:

Proline Diproline Triproline Tetraproline Pentaproline Hexaproline Ri~ 0.44

o.70

0.85

0.9o

0.7o

0.50

I t will be observed that for peptides higher than the tetramer the RF values decrease, and from heptaproline and above the RE values become smaller than that of the monomer. Under the conditions described above, adenylic acid gives a brown spot whose mobility is characterized b y an RF of 0.30. For quantitative determination of peptide contents the spots were extracted with 75 % ethanol and the color intensity was determined in a Klett photometer with a green filter (540 m/~). A certain amount of overlapping could not be avoided. Several runs were checked by fractional precipitation of higher peptides and determination of the molecular weights b y the ultracentrifuge and anhydrous titration. I t was found that the experimental error does not exceed 20 %. * S. ROGOZINSKI, p r i v a t e communication.

Biochim. Biophys. Acta, 14o (1967) 14-23

16

M. PAECHT-HOROWITZ, A. KATCHALSKY

Determination of molecular weights : Molecular weights were determined b y Mr. A. LUSTIG of the Department of Biophysics of this Institute with a Beckman analytical ultracentrifuge, model E, with a multichannel cell, according to YPHANTIS5. Equilibrium measurements were carried out on 1 % solutions and at an ionic strength of 0.2 at 29500 rev./min. End-group determination was carried out b y anhydrous titration of the free imino group with HC104 in glacial acetic acid 6 or by the titration of the free carboxylic groups with methoxide 7. The second method gave more reproducible results for higher polypeptides.

Synthetic j~roeesses Pre]baration of ibroline adenylate. The synthesis of proline adenylate followed the method of MICHELSON AND LETTERSs for glycine adenylate. The carbobenzoxy derivative of proline adenylate was subjected to catalytic hydrogenation to yield relatively pure active phosphate. CarbobenzoxyproIine was prepared according to BERGMANN AND ZERVAS9 as follows: 23 g proline were dissolved in warm 2 M NaOH. The solution was cooled and 34 g carbobenzoxy chloride were slowly added with vigorous stirring and with simultaneous addition of 50 ml neutralizing 4 M NaOH. The addition of reagents required about 35 min with constant cooling of the reaction mixture. Stirring at room temperature was continued for another IO rain and the solution extracted three times with Ioo-ml portions of ether. The aqueous solution was then cooled in an ice bath and 6 M HC1 added to lower the pH to the transition point of congo red. Carbobenzoxyproline precipitated as a heavy oil which was first washed with small amounts of cold water and then with a mixture of water and methanol. To obtain a sufficiently dry substance, as required for further manipulation, the carbobenzoxyproline was first dried in a desiccator overnight, then dissolved in absolute dioxane and freezedried. The evaporation of dioxane removed traces of water so that after five dissolutions and evaporations a pure substance was obtained. Left overnight in a deepfreezer the oily carbobenzoxyproline crystallized. 35 g of product was obtained (yield 70 %). The melting point was 73 ° (literature 76° ) and the analytical data were the following: Found: C, 63.21; H, 5.93; N, 5.16 %; carbobenzoxy groups 99.4. Calc.: C, 62.8; H, 6.03; N, 5.65 %; carbobenzoxy groups IOO.O. The small discrepancy between experimental and calculated seems to be due to traces of occluded dioxane. Carbobenzoxylbroline adenylate. 528 mg carbobenzoxyproline were dissolved in 2 ml absolute dioxane containing 0.72 ml tri-n-butylamine. The solution was cooled in an ice-bath, 0.22 ml chloroformate was added under cooling and the mixture left in the refrigerator for I h. During the cooling 229 mg adenylic acid was suspended in a mixture of 0.5 ml dimethylformamide 2blus 1.5 ml absolute dioxane containing 230 mg trioctylamine. The cooled solution of carbobenzoxyproline was then poured over the suspension of adenylic acid and the mixture was left overnight under anhydrous conditions and vigorous stirring. The next day the mixture was poured into IOO ml absolute diethyl ether, the precipitate centrifuged off, washed three times with small amounts of dry acetone and dried in a desiccator. The yield of carbobenzoxyproline adenylate was about 40 % of a material of 70-80 % purity. Since the washing with ether removed practically all of the unreacted carbobenzoxyproline, the major Biochim. Biophys. Acta, 14o (1967) 14-23

17

POLYMERIZATION OF PROLINE ADENYLATE

contaminant was an excess of adenylic acid which was removed in the later stages of the synthesis. Carbobenzoxyproline may be stored in dark bottles at --20 °. Analysis: hydroxamic acid test: 74 % (referred to carbobenzoxyproline benzyl ester). Spectroscopic measurements of carbobenzoxy groups (at 257-5 m~): 88 %. Phosphorus: calc., 5.39 % ; found 6.08 %. Melting point : 14°° (decomposition).

0

OH

,¢"--( I

OH

~2

"."

I

OH

Proline adenylate. I mM of carbobenzoxyproline adenylate, dissolved in 95 % acetic acid was hydrogenated over I g wet palladium black for 13-15 min. During the hydrogenation the solution is kept at o ° and the reaction mixture treated in a cold room. The catalyst is quickly filtered off, washed several times with ice-cold 9° % acetic acid and the total filtrate lyophilized immediately. Analysis: Hydroxamic acid test (referred to proline ethyl ester): 7 ° %. Benzyl groups (in order to check whether hydrogenation was complete), 5-1o %. Elementary analysis:Found: C, 41; H, 4.73; N, 18.9; P, 7.0%. Calc.: C, 41.5; H, 4.85; N, 18.5; P, 7.3 %. Chromatography in acid medium gave only one ninhydrin-positive spot. The hydrolytic behavior of the substance indicated that it was uniform. Polycondensation of proline adenylate at constant ~bhosphoanhydride concentration Proline adenylate was dissolved in 3 ml of dilute sodium carbonate buffer (approx. 0.05 M) at pH 8.5 to give a concentration of 23 mM. Since higher buffer concentrations increase the rate of hydrolysis, buffer concentration was kept as low as compatible with the maintenance of a constant pH. With the advancement of polycondensation, both the contents of anhydride and the pH decrease. To secure a state of constant phosphoanhydride concentration cc

5

y

~2 o

0~

o

0.6

O2

0

6

12

18

2~

30

t

(rain)

36

4~

4~

Fig. I. Time dependence of the added 0.3 M proline adenylate required to m a i n t a i n the concent r a t i o n of p h o s p h o a n h y d r i d e c o n s t a n t at 2 3 mM during the polycondensation (for details see text).

Biochim. Biophys. Acta, 14o (1967) 14-23

18

M. PAECHT-HOROWITZ, A. KATCHALSKY

and constant pH, o.I-ml test samples were withdrawn from the solution every few minutes for anhydride and pH determinations. Consequently, the reaction mixture was supplemented by appropriate amounts of 0.3 M proline adenylate and the pH adjusted to its initial value by dropwise addition of 0.5 M carbonate solution. Since the amounts withdrawn for testing exceeded those added to maintain constant phosphoanhydride concentration and constant pH, the volume of the reaction mixture decreases with time. Fig. I represents graphically the total amounts of proline adenylate added to the polymerization mixture during the reaction. As follows from the theory, the amounts increase with the advancement of the process. The samples taken during the experiment were chromatographed and the amount of peptides formed was recorded to give the kinetic runs of peptide formation. To check upon the distribution of molecular weights some runs were carried out at higher concentrations and the peptides obtained fractionated by acetone precipitation. The molecular weights of the peptides were determined ultracentrifugally and the corresponding RF values of the peptides established. Another series of polymerization experiments was carried out in the presence of an excess of free proline at room temperature and pH 8.5. The proline concentration was o.16 M while the concentration of proline adenylate was 15 mM. RESULTS AND DISCUSSION Fig. 2 represents the increase in the amount of various peptides with time. It will be observed that the monomer content (At) increases more rapidly than that of the higher polymers and in about 4° min it reaches a steady concentration which remains practically constant in all subsequent determinations. The growth of dipeptide contents (A2) under the present experimental conditions is slower and reaches 28

,

~

,

,

,

,

~ o ~

26

24

==

/S

I

,

°"

.- rno[e " l . rain "l )

f

18

~

16

a o~'~

. '

'

~

..

Az (Kz = 5"4° [" rn°le''' rain" }

m

b ,~ I 0

I

/ /

4

.

7

81- /

/

~

J ~.

/

. . . . . . . . ~ o l e "

/o

/ •

~..~"

,~.-.'~

~

.

~

. rain"l )

,

A4,Ka=IEL7, . . . . . ".rain "l ,

•

t (rain)

Fig. 2. P a t t e r n of p e p t i d e f o r m a t i o n as f u n c t i o n of time, in a p o l y m e r i z a t i o n process a t c o n s t a n t phosphoanhydride concentration.

Biochim. Biophys. Acta, 14o (1967) 14-23

POLYMERIZATION

OF PROLINE

19

ADENYLATE

2: 22 21:

20' 19

dA] Idt

18

17 16 15 14 13

-o

•

~

~

dA2 Idt

dA3/

2

a

o

I"( min} F i g . 3. R a t e of p e p t i d e f o r m a t i o n a s f u n c t i o n of t i m e . ( T h e t r i m e r s a n d t e t r a m e r s d e v i a t e c o n s i d e r ably from the theoretical curve, probably due to the difficulty in separating one from the other by paper chromatography.)

d~ dt

1 AP

1.5

,id2

20

2.5

3b

A~. ~0~

F i g . 4. T h e r a t e o f m o n o m e r f o r m a t i o n n o r m a l i z e d t o A P 0, a s f u n c t i o n of m o n o m e r c o n c e n t r a t i o n . T h e i n t e r c e p t kh = o . i m i n -1, f r o m t h e s l o p e f o l l o w s t h a t k I = 3-7 l - m o l e - 1 . m i n -1.

Biochim. Biophys. Acta, 1 4 o ( I 9 6 7 ) 1 4 - 2 3

20

M. PAECHT-HOROWITZ, A. KATCHALSKY

a steady-state value in about 5o rain. A sfmilar pattern is followed by other peptides, the tripeptide reaching a stationary value in about 60 min, etc. As shown theoretically, the stationary values of peptide concentration are independent of initial concentration and given b y the simple relations (as derived from Part I, Eqn. 41). A/(oo)

=

kh/ki

(i)

In our case the values for A/were the following:

Al(OO)

A2(~)

A3(cxD)

A4(oo)

27. 3 mY[

18.4 m M

11. 3 m M

9.3 m M

Thus for known kh (and, as will be shown in the next paragraph, its value is o.I rain -1) we can readily obtain all the k,'s from the stationary-state peptide concentration. The results evaluated in this manner are the following: D e g r e e of p o l y m e r i z a t i o n k ~ ( l . m o l e - l ' m i n -1)

i

2

kl ~ 3.65

3

k3 =

k2 = 5.4 °

4 8.80

k4 = lO.7

The values of dA,/dt, obtained graphically from Fig. 2, are plotted in Fig. 3. Now according to the theory, dA1/dl = k h . A P - - k l . A P ' A 1

(2)

Since AP = A P ( o ) = AP 0 = constant, the plot of (I/AP0) (dA1/dt) vs. A 1 should be a straight line with an intercept kh and a slope - - k 1. This conclusion was verified _d Lrl A2 dt

[ dA1 + dA2.~

1

I AP

T0-2

b

o

o

o

Q9

1.0

1.1

12

t3

1.4 A2.1(j2

1.5

1.6

1,7

i!8

o

,

1.4

IB

22

2.6 3.0 A1/A2

o

o

3,4

3.8

I

4.2

Fig. 5. a. The s u m of the rates of m o n o m e r a n d dimer formation, normalized to A P 0, vs. dimer concentration, b. The rate of change of the logarithm of dimer concentration vs. the ratio of m o n o m e r to dimer concentration.

Biochim. Biophys. Acta, 14o (1967) 14-23

POLYMERIZATION OF PROLINE ADENYLATE

21

by the evaluation of kh and k 1 from Fig. 4. The values of higher propagation constants may be evaluated in a similar manner. Thus I

dAs

APo

dt

-

kl'A1 -- ks'As

-

(3)

upon adding Eqns. 2 and 3

or

I

~dal

dA2~

A f% ~ T

+ ~ 7 1 = kh

-

ks-A2

(4)

Thus a plot of (I/AP0) (dA1/dt + dAjdt) v s . A s should give again a straight line with kh as the intercept and --k s as the slope. Alternatively Eqn. 3 may be r e w r i t t e n as

d In A3 dl 8

I

d In A s

AP0

dt

A1 -- kiT--2~s

I AP

0 6

/

4 2

ks

(5)

,/

/

o

-4

-6

-8

AzlA3

dt

dt

Of

Of

o

o

b

A 4 JO ~3

Fig. 6. a. T h e r a t e of c h a n g e of t h e l o g a r i t h m of t r i m e r c o n c e n t r a t i o n vs. t h e ratio of d i m e r to t r i m e r c o n c e n t r a t i o n , b. T h e s u m of t h e r a t e s of m o n o m e r , dimer, t r i m e r a n d t e t r a m e r f o r m a t i o n , vs. t e t r a m e r concentration. Biochim. Biophys. Acta, 14o (1967) 14-23

22

M. PAECHT-HOROWITZ, A. KATCHALSKY

which allows the evaluation of kl and k s. The results are given in Figs. 5a and b. In a general way we m a y write for any degree of polymerization I d In A,-1 AP0 dt

hi-1 A,-1

h,

(6)

or t

d XAk I

k=0

APo

-

dt

hh

-

-

-

k~. Al

(7)

Figs. 6a and b represent the graphs from which k s and k 4 were evaluated. The results of the kinetic evaluation of the rate constants as compared with the steady-state values are given in Table I. TABLE I Steady state

hh (min)

kl(l'mole-l" rain -1)

k2(l'mole -1" ks(l'mole-l" min -1) rain-1)

k4(l'mole-l" min -1)

by Eqn. I by Eqn. 6

o.Io

3.65 3.26

5.4 6.1

IO.I 11.9

8.8 9.1

The overall agreement of the results is satisfactory and allows the utilization of the k,'s for further calculation. As shown in Part I (Eqn. 40) the amount of peptide as function of time is given b y the following equation kh(

' e-~Ap0t/~,

j=l

kz

z=~ k ~

)

(s)

where l assumes the values from I to i, except the value j. In order to verify the theoretical prediction, a computer program was set up and the amount of A, evaluated for various times. The program was carried out by ,

i

120

I00

80

h

\

60

4O

2O

% |(min)

Fig. 7. The formation of various peptides with time, in the presence of an excess of monomer. Biochim. Biophys. Acta, 14o (1967) 14-23

POLYMERIZATION OF PROLINE ADENYLATE

23

the Fortran method, on a 16o4 A computer of the Control Data Corporation, U.S.A. The results of the calculation are plotted as the solid curves of Fig. 2. When free proline is added to the reaction mixture, theory predicts that the concentrations of the peptides will change with time according to Eqn. 41 of Part I. A~

(k,/k~) {I - -

' 2~ n=l

e- ' F

[I + knAl(O)/kh] / /~t [kj/(k 1 ;T=I

kn)] }

(9)

A typical run of A, as function of time is given in Fig. 7 which represents the change in monomer and polymer contents under steady-state conditions. The theoretical requirement is that whatever the initial value of the added free component, the stationary value should depend only on kh and k,. Indeed, a comparison of A,(oo) in Figs. 7 and 2 shows the value to be identical within experimental error. On the other hand, the details of the curves differ appreciably. Thus the rate of approach of monomer concentration to its steady-state value is significantly different when no proline was added or in the presence of a high excess of proline as shown in Fig. 8. dAi 1p 16~

dt

5

-5

-10 ~15, -20 -25 -30

0

10

20

a0

t(rain)

4b

10

Fig. 8. T h e r a t e of c h a n g e in t h e c o n c e n t r a t i o n of m o n o m e r , w h e n n o free m o n o m e r is p r e s e n t (0--0) a n d w h e n a n e x c e s s of m o n o m e r is p r e s e n t ( 0 - - 0 ) . ACKNOWLEDGEMENT

This investigation was supported by the U.S. Public Health Service, Research Grant GM-o7852-o 5, from the Division of General Medical Sciences. REFERENCES i 2 3 4 5 6 7 8 9

F. LIPMANN AND L. C. TUTTLE, jr. Biol. Chem., 159 (1945) 21. S. HESTRIN, J. Biol. Chem., 18o (1949) 249. J- B. SUMNER, Science, ioo (1944) 413 . W. TROLL AND J. LINDSLEY, J. Biol. Chem., 215 (1955) 655. D. A. YPHANTIS, Ann. N . Y . Aead. Sci., 88 (196o) 586. G. TO~NNIES AND T. P. CALLAN, jr. Biol. Chem., 125 (1938) 259. J- S. FRITZ AND N. M. LISlCKI, Anal. Chem., 23 (1951) 589 . A. M. MICHELSON AND R. LETTERS, Biochim. Biophys. Acta, 80 (1964) 242. M. BERGMANN AND L. ZERVAS, Chem. Ber., 65 (1932) 1192.

Biochim. Biophys. Acta, 14o (1967) 14,23