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On the binding of condensed phosphates by proteins
174 BBA
SHORT COMMUNICATIONS 43 oh9
On the binding of condensed phosphates by proteins Although strong interactions between condensed phosphates and proteins have been known for m a n y years a-a, little has been reported on the relationship between chain length of the phosphate and the extent of the interaction. It is known that the monomer, orthophosphate, interacts with bovine serum albumin only to a minor extent in competition with a monovalent dyO. In fact, orthophosphate interacts less strongly with this protein than do chloride or nitrate ions 5 in contrast to the strong interactions reported for condensed phosphate. In light of the interest in proteinphosphate interactions in the modification of milk, meat and various vegetable proteins and of the general chemical interest of the phenomenon of polyanionpolycation interactions, we have studied the interaction with two proteins of a variety of condensed phosphates. Gelatin (from General Foods Corp.) and egg-white solids (from Swift and Co.) were the proteins selected. To avoid variations due to aggregation of the gelatin, solutions of this protein were prepared in a reproducible manner as to warming to achieve solution and as to time held in solution (IO min) prior to running the procedure given below. The egg albumin was dissolved in water in I-1 batches of stock solution at o.5 % (w/w). The various phosphates were available from this laboratory. Other chemicals were of standard reagent grade. The method of study is based on the assumption that any interaction between a condensed phosphate and a protein molecule involves the same sites on the phosphate as are engaged in the formation of soluble calcium complexes with the phosphate. Thus any reduction in sequestration power in the presence of protein m a y be attributed to protein-phosphate interactions. The experimental procedure makes use of the semi-automatic nephelometer developed and used by IRANI AND CALLIS6. The protein was dissolved in water (clarified by filtering if necessary), the polyphosphate put in solution, and finally the competing precipitant was added. To this system was added, shot-by-shot, a solution of Ca(NO3)2 until a turbid endpoint was reached as detected by the nephelometer. Careful pH control was exercised. Temperature was that of the laboratory (28 ~ I°). For the preliminary work reported here, orthophosphate was selected as the precipitant. The amount of precipitant was adjusted to give a permanent turbidity when 1-2 drops of calcium solution were added in the absence of polyphosphate and protein. Binding of calcium by the protein was found to be negligible by test in competition with the precipitant. Precipitation of the orthophosphate alone in solution by calcium ion showed that the precipitate was hydroxylapatite at pH 7 and 9The endpoints with the long-chain phosphates were often difficult to ascertain to closer than ± 5 %. To obtain more useful results, the raw data were smoothed by multiple stepwise regression analysis on an IBM 7o4 digital computer and additional data points were interpolated from the resulting equations. Runs were made at two pH's, two protein concentrations, two polyphosphate concentrations and with five sodium polyphosphate species of average chain lengths 2, 3, 6, 12 and 5o. (The pyro- and triphosphate are purified crystalline species; the longer-chain phosphates are glasses containing a distribution of linear species.) The results shown in Figs. I - 4 are expressed as percentage reduction in seBiochim. Biophys. dcta, 12o (1966) I74-I76
175
SHORT COMMUNICATIONS
questering power versus chain length for the several conditions. The pyrophosphate systems always show a strong effect. For the others, there are effects which vary in intensity depending on the experiment. Distinct maxima and minima are present.
50
.<
I,..,
I t,.
|
40
o Q.
0 O_
== ":" 30 q)
30 ¢o
g ZO
¢1) (n
.E
.c g
g U
20
I0
\"._.. s/
@
(r
0
0
Phosphote Choin Length - ~ (log)
3
6
13
50
Phosphote Chnin Length-'~ (log)
F i g . I. R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y e g g w h i t e a t p H 7.0. - - - , 1 . o 2 - i o - 4 1% i n phosphorus; 5 mg/ml protein. ------, I.O2- lO -4 1% i n p h o s p h o r u s ; 2.5 m g / m l p r o t e i n . - - - - 2 . o 4- lO - 4 N i n p h o s p h o r u s ; 5 m g / r n l p r o t e i n . - . . . . . , 2 . o 4 . lO -4 1% i n p h o s p h o r u s ; 2. 5 m g / I n l protein. F i g . 2. R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y g e l a t i n a t p H 7.o. - - , 1 . o 2 - lO -4 1% i n p h o s p h o r u s ; 5 . o m g / m l p r o t e i n . - - - - - - , 1 . o 2 . lO -4 N i n p h o s p h o r u s ; 2.5 m g / m l p r o t e i n . - - - - 2 . 0 4 " lO -4 1% i n p h o s p h o r u s ; 5 . 0 m g / m l p r o t e i n . - . . . . , 2 . 0 4 - lO -4 N i n p h o s p h o r u s ; 2. 5 m g / m ] protein.
#u
#
40
40
o
50
30
g= 20
g
.s =
\
0
lI
g g Q2
~,, o
"'-" 3
20
®
,, I0
\
.c_
I0
%
,,
g
"~.
. 6
. 113 .
Phosphote Choin Length
. - ~
50
(log)
g (]~
%
o
,
Phosphote Choin Length - ~ (log)
F i g . 3- R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y i n t e r a c t i o n w i t h e g g w h i t e a t p H 9 . 0 . - - 1 . o 2 . lO -4 N i n p h o s p h o r u s ; 5 . 0 m g / m l p r o t e i n . - - - - - , I.O2. lO - 4 N i n p h o s p h o r u s ; 2.5 m g / r n l protein. , 2 . 0 4 . lO - 4 N i n p h o s p h o r u s ; 5 . 0 m g / m l p r o t e i n . - . . . . , 2 . 0 4 - lO -4 N i n p h o s p h o r u s ; 2.5 m g / m l p r o t e i n . F i g . 4. R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y g e l a t i n a t p H 9 . o . - - . , 1 . o 2 . lO -4 N i n phosphorus; 5.0 mg/ml protein. , 1 . o 2 . lO -4 1~ i n p h o s p h o r u s ; 2. 5 m g / m l p r o t e i n . - - - - 2 . o 4 . lO -4 1% i n p h o s p h o r u s ; 5 . 0 m g / r n l p r o t e i n . - . . . . . , 2 , o 4- lO -4 N i n p h o s p h o r u s ; 2.5 m g / m l protein.
Biochim. Biophys. Acta, 12o (1966) 1 7 4 - 1 7 6
176
SHORT COMMUNICATIONS
BRIGGS3 has shown t h a t condensed phosphates interact with the cationic sites on proteins and t h a t there is behavior consistent with the laws of mass action. The b i n d i n g in this s t u d y is p r o b a b l y with lysine a n d arginine sitesT, 8. The present results show a mass action effect - b i n d i n g increases with increase in protein at a given polyphosphate concentration, for example. There is not enough i n f o r m a t i o n on the d a t a to allow a selection between b i n d i n g modes: site b i n d i n g or diffuse electrostatic b i n d i n g9. Nor, in fact, can we be sure of the n u m b e r of dissociable sites on, say, the phosphate which are involved per interaction since this is k n o w n to v a r y even in b i n d i n g of simple counterions6. Not unexpectedly, there is little difference in b i n d i n g at p H 7 a n d p H 9- This is the region where the a c i d - b a s e t i t r a t i o n curves for the proteins are nearly flat~°, al a n d the h y d r o d y n a m i c shape factors are essentially c o n s t a n t ~2. There is clearly an effect due to chain length of the polyphosphate; the observed effects should be qualified b y the observation t h a t a protein interaction involving, for example, one charged site on a pyrophosphate molecule will affect its ability to complex calcium ion m u c h more t h a n the same interaction will affect triphosphate. I n the latter case, if the interaction be at one end of the triphosphate, a pyrophosphate " t a i l " remains which m a v be able to form m o d e r a t e l y stable calcium complexes. This is in contrast to the situation for the pyrophosphate, where one interaction with a protein site might well eliminate almost t o t a l l y its complexing power for calcium. For the long-chain phosphate species there are p r o b a b l y multiple interactions with the proteins. The overall effect in terms of calcium complex formation will depend on the fraction of sites not b o u n d to the protein a n d on the a r r a n g e m e n t of these "free" sites. F u r t h e r s t u d y of this kind of competitive p h e n o m e n o n should lead to detailed i n f o r m a t i o n as to site spacing on the protein a n d shape a n d conformation factors. Research Departme.J#, Inorganic Chemicals Division, Monsanto Company, St. Louis, Mo. (U.S.A.)
JOHN W. LYONS CHARLES D. SIEBENTHAL*
I H. HERRMANNAND G. E. PERLMANN, Nature, 14o (I936) 807. Biochem. J., 32 (19381 926. 3 p. R. BRIGGS,J. Biol. Chem., 134 (194o) 261. 4 F. KARUSH, J. Am. Chem. Soc., 73 (1951) 1246. 5 I. M. KLOTZ AND J. M. URQUHART, J. Phys. Colloid Chem., 53 (1949) IOO. 0 R. R. IRANI AND C. F. CALLIS, J. Phys. Chem., 64 (t96o) 1398. 7 R. A. ALBERTY, in H. •EURATH AND K. BAILEY, The Proteins, Vol. IA, Academic Press, New York, 1953. 8 I.M. KLOTZ,in H. NEURATHAND ~X~.BAILEY, The Proteins, Vol. IB, Academic Press, New York, 1953. 9 J- Vq. LYONSAND L. KOTIN,J. Am. Chem. Soc., 87 (t965) 167o. io A. W. KENCHINGTONAND A. G. WARD, Biochem. J., 58 (I954) 202. I I t { . K. CANNAN, A. KIBRICKAND A. H. PALMER,Ann. N.Y. Aead. Sci., 41 (1941) 243. 12 P. A. CHARLWOODAND A. ENS, Can. J. Chem., 35 (1957) 99. 2 G. E. PERLMANN AND H . HERRMANN,
Received J u n e 2nd, 1965 Revised m a n u s c r i p t received N o v e m b e r 4th, 1965 * Present address: Shell Development Company, Emeryville, Calif. (U.S.A.). Biochim. Biophys..qcta, i2o (I966) I74-I70
SHORT COMMUNICATIONS 43 oh9
On the binding of condensed phosphates by proteins Although strong interactions between condensed phosphates and proteins have been known for m a n y years a-a, little has been reported on the relationship between chain length of the phosphate and the extent of the interaction. It is known that the monomer, orthophosphate, interacts with bovine serum albumin only to a minor extent in competition with a monovalent dyO. In fact, orthophosphate interacts less strongly with this protein than do chloride or nitrate ions 5 in contrast to the strong interactions reported for condensed phosphate. In light of the interest in proteinphosphate interactions in the modification of milk, meat and various vegetable proteins and of the general chemical interest of the phenomenon of polyanionpolycation interactions, we have studied the interaction with two proteins of a variety of condensed phosphates. Gelatin (from General Foods Corp.) and egg-white solids (from Swift and Co.) were the proteins selected. To avoid variations due to aggregation of the gelatin, solutions of this protein were prepared in a reproducible manner as to warming to achieve solution and as to time held in solution (IO min) prior to running the procedure given below. The egg albumin was dissolved in water in I-1 batches of stock solution at o.5 % (w/w). The various phosphates were available from this laboratory. Other chemicals were of standard reagent grade. The method of study is based on the assumption that any interaction between a condensed phosphate and a protein molecule involves the same sites on the phosphate as are engaged in the formation of soluble calcium complexes with the phosphate. Thus any reduction in sequestration power in the presence of protein m a y be attributed to protein-phosphate interactions. The experimental procedure makes use of the semi-automatic nephelometer developed and used by IRANI AND CALLIS6. The protein was dissolved in water (clarified by filtering if necessary), the polyphosphate put in solution, and finally the competing precipitant was added. To this system was added, shot-by-shot, a solution of Ca(NO3)2 until a turbid endpoint was reached as detected by the nephelometer. Careful pH control was exercised. Temperature was that of the laboratory (28 ~ I°). For the preliminary work reported here, orthophosphate was selected as the precipitant. The amount of precipitant was adjusted to give a permanent turbidity when 1-2 drops of calcium solution were added in the absence of polyphosphate and protein. Binding of calcium by the protein was found to be negligible by test in competition with the precipitant. Precipitation of the orthophosphate alone in solution by calcium ion showed that the precipitate was hydroxylapatite at pH 7 and 9The endpoints with the long-chain phosphates were often difficult to ascertain to closer than ± 5 %. To obtain more useful results, the raw data were smoothed by multiple stepwise regression analysis on an IBM 7o4 digital computer and additional data points were interpolated from the resulting equations. Runs were made at two pH's, two protein concentrations, two polyphosphate concentrations and with five sodium polyphosphate species of average chain lengths 2, 3, 6, 12 and 5o. (The pyro- and triphosphate are purified crystalline species; the longer-chain phosphates are glasses containing a distribution of linear species.) The results shown in Figs. I - 4 are expressed as percentage reduction in seBiochim. Biophys. dcta, 12o (1966) I74-I76
175
SHORT COMMUNICATIONS
questering power versus chain length for the several conditions. The pyrophosphate systems always show a strong effect. For the others, there are effects which vary in intensity depending on the experiment. Distinct maxima and minima are present.
50
.<
I,..,
I t,.
|
40
o Q.
0 O_
== ":" 30 q)
30 ¢o
g ZO
¢1) (n
.E
.c g
g U
20
I0
\"._.. s/
@
(r
0
0
Phosphote Choin Length - ~ (log)
3
6
13
50
Phosphote Chnin Length-'~ (log)
F i g . I. R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y e g g w h i t e a t p H 7.0. - - - , 1 . o 2 - i o - 4 1% i n phosphorus; 5 mg/ml protein. ------, I.O2- lO -4 1% i n p h o s p h o r u s ; 2.5 m g / m l p r o t e i n . - - - - 2 . o 4- lO - 4 N i n p h o s p h o r u s ; 5 m g / r n l p r o t e i n . - . . . . . , 2 . o 4 . lO -4 1% i n p h o s p h o r u s ; 2. 5 m g / I n l protein. F i g . 2. R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y g e l a t i n a t p H 7.o. - - , 1 . o 2 - lO -4 1% i n p h o s p h o r u s ; 5 . o m g / m l p r o t e i n . - - - - - - , 1 . o 2 . lO -4 N i n p h o s p h o r u s ; 2.5 m g / m l p r o t e i n . - - - - 2 . 0 4 " lO -4 1% i n p h o s p h o r u s ; 5 . 0 m g / m l p r o t e i n . - . . . . , 2 . 0 4 - lO -4 N i n p h o s p h o r u s ; 2. 5 m g / m ] protein.
#u
#
40
40
o
50
30
g= 20
g
.s =
\
0
lI
g g Q2
~,, o
"'-" 3
20
®
,, I0
\
.c_
I0
%
,,
g
"~.
. 6
. 113 .
Phosphote Choin Length
. - ~
50
(log)
g (]~
%
o
,
Phosphote Choin Length - ~ (log)
F i g . 3- R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y i n t e r a c t i o n w i t h e g g w h i t e a t p H 9 . 0 . - - 1 . o 2 . lO -4 N i n p h o s p h o r u s ; 5 . 0 m g / m l p r o t e i n . - - - - - , I.O2. lO - 4 N i n p h o s p h o r u s ; 2.5 m g / r n l protein. , 2 . 0 4 . lO - 4 N i n p h o s p h o r u s ; 5 . 0 m g / m l p r o t e i n . - . . . . , 2 . 0 4 - lO -4 N i n p h o s p h o r u s ; 2.5 m g / m l p r o t e i n . F i g . 4. R e d u c t i o n i n s e q u e s t e r i n g p o w e r c a u s e d b y g e l a t i n a t p H 9 . o . - - . , 1 . o 2 . lO -4 N i n phosphorus; 5.0 mg/ml protein. , 1 . o 2 . lO -4 1~ i n p h o s p h o r u s ; 2. 5 m g / m l p r o t e i n . - - - - 2 . o 4 . lO -4 1% i n p h o s p h o r u s ; 5 . 0 m g / r n l p r o t e i n . - . . . . . , 2 , o 4- lO -4 N i n p h o s p h o r u s ; 2.5 m g / m l protein.
Biochim. Biophys. Acta, 12o (1966) 1 7 4 - 1 7 6
176
SHORT COMMUNICATIONS
BRIGGS3 has shown t h a t condensed phosphates interact with the cationic sites on proteins and t h a t there is behavior consistent with the laws of mass action. The b i n d i n g in this s t u d y is p r o b a b l y with lysine a n d arginine sitesT, 8. The present results show a mass action effect - b i n d i n g increases with increase in protein at a given polyphosphate concentration, for example. There is not enough i n f o r m a t i o n on the d a t a to allow a selection between b i n d i n g modes: site b i n d i n g or diffuse electrostatic b i n d i n g9. Nor, in fact, can we be sure of the n u m b e r of dissociable sites on, say, the phosphate which are involved per interaction since this is k n o w n to v a r y even in b i n d i n g of simple counterions6. Not unexpectedly, there is little difference in b i n d i n g at p H 7 a n d p H 9- This is the region where the a c i d - b a s e t i t r a t i o n curves for the proteins are nearly flat~°, al a n d the h y d r o d y n a m i c shape factors are essentially c o n s t a n t ~2. There is clearly an effect due to chain length of the polyphosphate; the observed effects should be qualified b y the observation t h a t a protein interaction involving, for example, one charged site on a pyrophosphate molecule will affect its ability to complex calcium ion m u c h more t h a n the same interaction will affect triphosphate. I n the latter case, if the interaction be at one end of the triphosphate, a pyrophosphate " t a i l " remains which m a v be able to form m o d e r a t e l y stable calcium complexes. This is in contrast to the situation for the pyrophosphate, where one interaction with a protein site might well eliminate almost t o t a l l y its complexing power for calcium. For the long-chain phosphate species there are p r o b a b l y multiple interactions with the proteins. The overall effect in terms of calcium complex formation will depend on the fraction of sites not b o u n d to the protein a n d on the a r r a n g e m e n t of these "free" sites. F u r t h e r s t u d y of this kind of competitive p h e n o m e n o n should lead to detailed i n f o r m a t i o n as to site spacing on the protein a n d shape a n d conformation factors. Research Departme.J#, Inorganic Chemicals Division, Monsanto Company, St. Louis, Mo. (U.S.A.)
JOHN W. LYONS CHARLES D. SIEBENTHAL*
I H. HERRMANNAND G. E. PERLMANN, Nature, 14o (I936) 807. Biochem. J., 32 (19381 926. 3 p. R. BRIGGS,J. Biol. Chem., 134 (194o) 261. 4 F. KARUSH, J. Am. Chem. Soc., 73 (1951) 1246. 5 I. M. KLOTZ AND J. M. URQUHART, J. Phys. Colloid Chem., 53 (1949) IOO. 0 R. R. IRANI AND C. F. CALLIS, J. Phys. Chem., 64 (t96o) 1398. 7 R. A. ALBERTY, in H. •EURATH AND K. BAILEY, The Proteins, Vol. IA, Academic Press, New York, 1953. 8 I.M. KLOTZ,in H. NEURATHAND ~X~.BAILEY, The Proteins, Vol. IB, Academic Press, New York, 1953. 9 J- Vq. LYONSAND L. KOTIN,J. Am. Chem. Soc., 87 (t965) 167o. io A. W. KENCHINGTONAND A. G. WARD, Biochem. J., 58 (I954) 202. I I t { . K. CANNAN, A. KIBRICKAND A. H. PALMER,Ann. N.Y. Aead. Sci., 41 (1941) 243. 12 P. A. CHARLWOODAND A. ENS, Can. J. Chem., 35 (1957) 99. 2 G. E. PERLMANN AND H . HERRMANN,
Received J u n e 2nd, 1965 Revised m a n u s c r i p t received N o v e m b e r 4th, 1965 * Present address: Shell Development Company, Emeryville, Calif. (U.S.A.). Biochim. Biophys..qcta, i2o (I966) I74-I70