275
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 2 7 5 - - 2 8 0 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27860
PROTEIN-POLYNUCLEOTIDE SCATTERING C E N T E R S AS A P R O T E I N S T R U C T U R E PROBE
M I L T O N E D D Y M c D O N N E L L * a n d J O H N W. P R E I S S
Department of Physics, University of Delaware, Newark, Del. 19711 (U.S.A.) (Received September 30th, 1975)
Summary When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of a b o u t 10 9 particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the a m o u n t of enzyme that remains biologically active after being heated.
Large, low density scattering centers of a b o u t 10 9 particle weight are produced from certain mixtures of basic globular proteins and polynucleotides [1,2]. For these giant scattering centers to be created, the ratio of sufficiently long nucleic acid fragments to proteins must be close to a critical value. If the a m o u n t of nucleic acid in the system is changed to shift the ratio away from the critical value, the scattering from the solution decreases. This leads to the paradoxical situation where the doubling of the nucleic acid concentration may decrease the scattering of the solution to a hundredth of its former value witho u t a precipitate forming. In this paper we describe the use of giant protein-nucleic acid light scattering centers to probe conformational changes of the enzymes bovine pancreatic ribonuclease and chicken lysozyme. Before mixing with polyribonucleotides, aqueous solutions of these enzymes are held at elevated temperatures for varying lengths of time. This treatment essentially divides the molecules into two classes: native, globular proteins and inactivated enzymes. The number of molecules in the native conformation is proportional to the specific enzymatic ac* P r e s e n t address: D e p a r t m e n t o f M a c r o m o l e c u l a r S c i e n c e , Case Western R e s e r v e U n i v e r s i t y , Cleveland, O h i o 44106, U.S.A.
276 tivity remaining in the solution, that is, the rate at which ribonuclease hydrolyzes ribonucleotides containing pyrimidine and the rate at which lysozyme digests bacterial cell wall substrate. Since thermal denaturation of a globular protein often leaves most of the conformation of the molecule unaffected, one might suspect identical interactions between nucleic acid and active or denatured proteins which do not have a biological activity related to nucleic acids. Comparisons between light scattering from giant complexes containing nucleic acid and either only native protein or both active and inactivated enzyme show that this is n o t the case. Deviations from native complexing properties are proportional to the decrease in protein activity; the scattering centers directly reflect the altered protein conformation. Enzymes and enzyme substrates were prepared by General Biochemicals and polynucleotides with weight average molecular weights greater than 10 s were obtained from Miles Laboratories. All were used without further purification in 0.1 M sodium acetate buffer at pH 5.4. Heat was chosen as the denaturing agent for these experiments so that no new chemical reactant had to be introduced which might be incorporated into the scattering centers in u n k n o w n ways. Also heat alters the conformation without disrupting the covalent bonds. Since very low concentrations of components are required to form the large protein-nucleic acid scattering centers, the concentration of enzymes which is heated can be small enough that aggregates of protein do n o t precipitate from the solution. Enzymes were thermally denatured at a concentration of 100 #g/ml in lots of 2.4 ml. Samples in closed 10-ml vials were placed in an aluminium heating block for the specified time and then suspended in a beaker to come to room temperature. The light scattering procedures used in these experiments have been described previously [1]. The biological activity of ribonuclease was measured by the m e t h o d of Anfinsen et al. [3], and that of lysozyme, by the procedure of Smolelis and Hartsell [4]. When solutions of ribonuclease or lysozyme are heated at temperature T for time t, the activity A of the solution after it is returned to room temperature has decreased exponentially to A = Ao e x p ( - K T t )
The decay constants K w at several temperatures are given in Table I. This rate of change holds until more than 99% of the molecules are inactive. Fig. 1 shows the scattered intensity of 15/zg/ml of ribonuclease mixed with various concentrations of poly (G) when the protein has previously been heated
TABLEI Enzy me
T (°C)
K T (rain -1)
Ribonuclease
120 100 80
0.055 0.0107 0.0030
Lysozyme
120 100
0.064 0.0083
277
10 4
~o 4.
10:
10 3-
G
102
lC
102-
0
I ~glrnl
I 10 p ol y ( G )
I
20
10
0
10
~g/ml
p oly(G)
Fig. 1. Scattered light i n t e n s i t y G a t 4 5 (filled) a n d 1 3 5 ° ( h o l l o w ) 15 m i n after mixing from v a r i a b l e [ p o l y ( G ) ] a n d [ R ] = 15 p g / m l . The ribonuclease has previously been heated at 1 2 0 ° C f o r 0 (cD, 15 (~)0 3 0 (D), or 6 0 rain (v). Fig. 2. S c a t t e r e d light i n t e n s i t y G at 4 5 (filled) a n d 1 3 5 ° ( h o l l o w ) 1 5 m i n a f t e r m i x i n g f r o m v a r i a b l e [poly (G)] and [ R ] = 15 (o), 7.5 (A), or 3 . 7 5 Mg/ml (~).
at 120°C for 0, 15, 30, or 60 min. Note that as the protein has been heated for a longer period of time, the maximum scatter occurs with a lower concentration of nucleic acid. This is reminiscent of the previous study which showed less nucleic acid was needed for maximum scatter when the active enzyme concentration was decreased by dilution of the protein rather than by denaturation [1]. Data from this contrasting experiment are shown in Fig. 2 where various concentrations of poly (G) are mixed with 15, 7.5, or 3.75 pg/ml of unheated ribonuclease. For the data of Fig. 2 there is a critical ratio of the concentration of nucleic acid to the concentration of protein. The circumstances for the experiments represented in Figs. 1 and 2 certainly are n o t identical because only in the present experiments {Fig. 1) does the mass concentration of inactive molecules increase as the concentration of active molecules decreases. These inactive molecules enter into the scattering centers to increase the magnitude of m a x i m u m G above the value for decreased concentrations of unheated protein. The ratio of concentrations of poly (G) to active ribonuclease needed to produce m a x i m u m scatter is no longer constant; however, it varies quite systematically with the length of time the enzyme has been heated prior to mixing with the nucleic acid. The critical concentration of poly (G) for maximum scattering decreases exponentially with the length of time the solution has been heated. Since both the activity and the critical h o m o p o l y m e r concentration decrease exponentially
20
278 20'
10
~ug/ml poIy~G)ctit
j
I
I lll~
t
j
~
J I ~'lllb
2
,u g/ml Ractive Fig. 3. [poly (G)]czJt" which gives m a x i m u m scattered light for different [Racllve]. Active zJbonuclease concentration is cut by heating at 120 (o). 100 (A), 80°C (~), or by dilution (
Biochimica et Biophysica Acta, 4 2 8 ( 1 9 7 6 ) 2 7 5 - - 2 8 0 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27860
PROTEIN-POLYNUCLEOTIDE SCATTERING C E N T E R S AS A P R O T E I N S T R U C T U R E PROBE
M I L T O N E D D Y M c D O N N E L L * a n d J O H N W. P R E I S S
Department of Physics, University of Delaware, Newark, Del. 19711 (U.S.A.) (Received September 30th, 1975)
Summary When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of a b o u t 10 9 particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the a m o u n t of enzyme that remains biologically active after being heated.
Large, low density scattering centers of a b o u t 10 9 particle weight are produced from certain mixtures of basic globular proteins and polynucleotides [1,2]. For these giant scattering centers to be created, the ratio of sufficiently long nucleic acid fragments to proteins must be close to a critical value. If the a m o u n t of nucleic acid in the system is changed to shift the ratio away from the critical value, the scattering from the solution decreases. This leads to the paradoxical situation where the doubling of the nucleic acid concentration may decrease the scattering of the solution to a hundredth of its former value witho u t a precipitate forming. In this paper we describe the use of giant protein-nucleic acid light scattering centers to probe conformational changes of the enzymes bovine pancreatic ribonuclease and chicken lysozyme. Before mixing with polyribonucleotides, aqueous solutions of these enzymes are held at elevated temperatures for varying lengths of time. This treatment essentially divides the molecules into two classes: native, globular proteins and inactivated enzymes. The number of molecules in the native conformation is proportional to the specific enzymatic ac* P r e s e n t address: D e p a r t m e n t o f M a c r o m o l e c u l a r S c i e n c e , Case Western R e s e r v e U n i v e r s i t y , Cleveland, O h i o 44106, U.S.A.
276 tivity remaining in the solution, that is, the rate at which ribonuclease hydrolyzes ribonucleotides containing pyrimidine and the rate at which lysozyme digests bacterial cell wall substrate. Since thermal denaturation of a globular protein often leaves most of the conformation of the molecule unaffected, one might suspect identical interactions between nucleic acid and active or denatured proteins which do not have a biological activity related to nucleic acids. Comparisons between light scattering from giant complexes containing nucleic acid and either only native protein or both active and inactivated enzyme show that this is n o t the case. Deviations from native complexing properties are proportional to the decrease in protein activity; the scattering centers directly reflect the altered protein conformation. Enzymes and enzyme substrates were prepared by General Biochemicals and polynucleotides with weight average molecular weights greater than 10 s were obtained from Miles Laboratories. All were used without further purification in 0.1 M sodium acetate buffer at pH 5.4. Heat was chosen as the denaturing agent for these experiments so that no new chemical reactant had to be introduced which might be incorporated into the scattering centers in u n k n o w n ways. Also heat alters the conformation without disrupting the covalent bonds. Since very low concentrations of components are required to form the large protein-nucleic acid scattering centers, the concentration of enzymes which is heated can be small enough that aggregates of protein do n o t precipitate from the solution. Enzymes were thermally denatured at a concentration of 100 #g/ml in lots of 2.4 ml. Samples in closed 10-ml vials were placed in an aluminium heating block for the specified time and then suspended in a beaker to come to room temperature. The light scattering procedures used in these experiments have been described previously [1]. The biological activity of ribonuclease was measured by the m e t h o d of Anfinsen et al. [3], and that of lysozyme, by the procedure of Smolelis and Hartsell [4]. When solutions of ribonuclease or lysozyme are heated at temperature T for time t, the activity A of the solution after it is returned to room temperature has decreased exponentially to A = Ao e x p ( - K T t )
The decay constants K w at several temperatures are given in Table I. This rate of change holds until more than 99% of the molecules are inactive. Fig. 1 shows the scattered intensity of 15/zg/ml of ribonuclease mixed with various concentrations of poly (G) when the protein has previously been heated
TABLEI Enzy me
T (°C)
K T (rain -1)
Ribonuclease
120 100 80
0.055 0.0107 0.0030
Lysozyme
120 100
0.064 0.0083
277
10 4
~o 4.
10:
10 3-
G
102
lC
102-
0
I ~glrnl
I 10 p ol y ( G )
I
20
10
0
10
~g/ml
p oly(G)
Fig. 1. Scattered light i n t e n s i t y G a t 4 5 (filled) a n d 1 3 5 ° ( h o l l o w ) 15 m i n after mixing from v a r i a b l e [ p o l y ( G ) ] a n d [ R ] = 15 p g / m l . The ribonuclease has previously been heated at 1 2 0 ° C f o r 0 (cD, 15 (~)0 3 0 (D), or 6 0 rain (v). Fig. 2. S c a t t e r e d light i n t e n s i t y G at 4 5 (filled) a n d 1 3 5 ° ( h o l l o w ) 1 5 m i n a f t e r m i x i n g f r o m v a r i a b l e [poly (G)] and [ R ] = 15 (o), 7.5 (A), or 3 . 7 5 Mg/ml (~).
at 120°C for 0, 15, 30, or 60 min. Note that as the protein has been heated for a longer period of time, the maximum scatter occurs with a lower concentration of nucleic acid. This is reminiscent of the previous study which showed less nucleic acid was needed for maximum scatter when the active enzyme concentration was decreased by dilution of the protein rather than by denaturation [1]. Data from this contrasting experiment are shown in Fig. 2 where various concentrations of poly (G) are mixed with 15, 7.5, or 3.75 pg/ml of unheated ribonuclease. For the data of Fig. 2 there is a critical ratio of the concentration of nucleic acid to the concentration of protein. The circumstances for the experiments represented in Figs. 1 and 2 certainly are n o t identical because only in the present experiments {Fig. 1) does the mass concentration of inactive molecules increase as the concentration of active molecules decreases. These inactive molecules enter into the scattering centers to increase the magnitude of m a x i m u m G above the value for decreased concentrations of unheated protein. The ratio of concentrations of poly (G) to active ribonuclease needed to produce m a x i m u m scatter is no longer constant; however, it varies quite systematically with the length of time the enzyme has been heated prior to mixing with the nucleic acid. The critical concentration of poly (G) for maximum scattering decreases exponentially with the length of time the solution has been heated. Since both the activity and the critical h o m o p o l y m e r concentration decrease exponentially
20
278 20'
10
~ug/ml poIy~G)ctit
j
I
I lll~
t
j
~
J I ~'lllb
2
,u g/ml Ractive Fig. 3. [poly (G)]czJt" which gives m a x i m u m scattered light for different [Racllve]. Active zJbonuclease concentration is cut by heating at 120 (o). 100 (A), 80°C (~), or by dilution (
