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Molecular aggregation of bovine pancreatic ribonuclease induced by substrate
.\lK!HIVES
OF
BIOCHEWISTI~Y
Molecular
AND
137, 373-378 (1970)
BIOPHYSICS
Aggregation
of Bovine
Induced
cli Chimica
delle Macromolecole Macromoldcwlaire Received
Ribonuclease
by Substrate
A. 1\IARZOTTO’ Isktuto
Pancreatic
L. GALZIGNA2
AND
(C.N.R.), Padova, Italy and the Dkpartement (CRS) BP 1018 34 Montpellier, France
September
30, 1969; accepted
January
de Biochimie
8, 1970
The interaction between bovine cytidine-2’,3’-cyclic phosphate pancreatic ribonllclease and ribonucleic acid or cytidine-2’,3’-cyclic phosphate is studied by means of t,he gel-filtration method. The formation of a molecular aggregate of the enzyme as a function of time is shown. Some _ physicochemical properties of the aggregated prod\lct _ have been investigated.
It is well known (1) that specific experimental conditions can induce the formation of molecular aggregates of ribonuclease (RNase A) such as dimers, tetramers, and polymers. This study stems from our previous observation concerning the interaction with substrate which appeared to induce an aggregation of RNase A as well. We studied thoroughly such a phenomenon by means of gel filtration (2), by taking ribonuclease samples reacted with both natural (RNA) and synthetic substrate (cytidine-2’) 3’cyclic phosphate) at variable hydrolysis times. MATERIALS
AND
METHODS
RNase A was prepared according to t,he Hirs procedure (3), from commercial bovine ribonuclease (Serovac Laboratories). BPN’ bacterial protease (Nagarse) was purchased by Nagase Co. Ltd., Osaka, Japan. RNase S’ was prepared as described by Marzot,to (4) by proteolytic digestion of RNase A with Nagarse using a 2O:l weight ratio in a 0.1 M KC1 solution at 2-3”. The pH was maintained at 8 by addition of a 0.1 M NaOH solution. After the addition of 1.6 equivalents of NaOH per mole of protein, the reaction was stopped with standard FlCl. RNasr S was recovered by chromatography ’ Present, Address: Laboratorio Chimica Tecnologic Itadio-elementi C.N.B ., Padova, It,aly. 2 Contribution No. 38 from t,he Research Group (INSIj:RM)
on a CG 50 Amberlite column (5 X 60 cm) eluted with 0.2 M phosphat,e buffer at p1-I G.5. S-protein and S-peptide were separated with a G-100 Sephadex column eluted with 50(% acetic acid and then lyophilized. The depolymerase activity determination of RNaseI A RNase S’, and molecular aggregates were carrfied out as reported by Marzotto (5) [Ising RNA as a substrate. Yeast ribonllcleic acid (RNA) from British Drug Houses Ltd. purified before the use as indicated elsewhere (6) and cytidine-2’,3’-cyclic phosphate from Koch-Light Laboratories were used as substrates. The molecular weight of RNA rarlged between 140,000 and 160,000 as determined by the gel-filtration method of Whitaker (2) and by light-scattering measurements using a Sofica L.S. photometer Model. 42,000, with cylindrical cells immersed in highly purified toluene. The instrument was standardized with benzene (clarified with accuracy) (Ising Rw (4360 A) = 48.5 X 10-j (7). The measurements were made in 0.1 ~RI acetate buffer pH 5.7 and 0.2 M NaCl aqueous solution at, X = 4360 8 using the value dn/dc = 0.198. All the solutions used were clarified by prolouged shaking with chloroform-isoamyl alcohol mixture, followed by centrifugation at 25,000g for 2 hr. RNA concent,rations were determined by spectrophotometric measurements of the optical density at 257 m/l. The range of RNA concentrat,ions used was between 1.5 X 1OF and 1.5 X 1OF g/ml. The concentrations of RNase A, RNaseL S’ > and S-protein were determined by spectrophotometric measurements at 280 rnp (S), and the concentrations of the molecrdar ag373
374
MARZOTTO
AND
gregate were determined on the basis of the phosphate content. The circular dichroism (CD) measurements in the far-ultraviolet were performed by means of a Roussel-Jouan (Paris) dichrograph at 25”. Cylindrical opticell quartz cells were used with 0.05 cm optical path. The sensitivity was kept at 1 X 1W5. The samples contained 1 mg of the molecular aggregate per milliliter of an aqueous solution. The molecular weight were also calculated from a calibration curve obtained with pure samples of a number of standard proteins. Experiments of protein aggregation were carried out in the presence of RNA in 0.1 M acetate buffer pH 5 and cytidine-2’,3’ cyclic phosphate in buffer pH 7 at 25”. The weight 0.1 M Tris-HCl ratio between protein and substrate was 1:15-20 with RNA as a substrate as I:2 with cytidine as a substrate. In a typical experiment 0.090 g of RNase A dissolved in 3 ml of 0.1 M acetate buffer (pH 5) were added to 6 ml of acetate solution containing 1.4 g of RNA. Molecular aggregation was measured at different times by withdrawing from the reaction mixture 3-ml samples and by passing them through a G-75 Sephadex (1.8 X 140 cm) column equilibrated and eluted with 0.1 M acetate buffer at pH 5. A preliminary elution of the enzyme alone in absence of RNA was also done. The molecular weights were calculated by means of a calibrat,ion curve established with various proteins of different molecular weight. The aggregation product was desalted on a G-25 Sephadex column (1.8 X 140 cm) eluted with 5% acetic acid and then lyophylized. RESULTS
Figure 1 reports a saturation curve obtained at a fixed concentration of natural S-peptide and varying amounts of S-protein. Fractional activity (a) is expressed as a percent of the maximum value observed. The shape of this curve was tentatively ascribed to an aggregation effect and stimulated a check of the molecular weights of S-protein and RNase S’ in order to see if they could be influenced by the presence of the substrate. The chromatographic behavior on G-75 Sephadex of RNase A, S-protein, and RNase S’ before and after their interaction with substrate is presented in Fig. 2. The colorimetric reaction with ninhydrin was quantitated by optical density measurements at 570 rnfi.
GALZIGNA
::[ /-----TV06L
/
0.4
02
:
I 2
-
““’ 5
FIG. 1. Initial velocities of RNA depolymerization (a) expressed as a percent of the maximum value observed are reported as a function of the molar ratio (T) between increasing amounts of S-protein and a constant concentration (5.24.10-7 M) of S-peptide.
Figure 2a shows an evident increase of the peak corresponding to the molecular aggregate between RNase A and RNA and a corresponding decrease of the peak relative to native RNase as a function of the reaction time. The ultraviolet spectrum of an aqueous solution (0.5 mg/l ml) of the aggregated product corresponding to the chromatographic fraction with elution volume of 260 ml is given in Fig. 3, which shows a maximum value of absorbancy at 257 rnp, typical of RNA nucleotides, and a shoulder at 277 rnp, likely due to phenolic residues present in the protein. The thermal transition curve for the aqueous solution containing 1 mg/ml of the RNase molecular aggregate obtained as indicated in Fig. 2a is reported in Fig. 4. [01]at 365 rnb is given as a function of temperature. The curve shows a double transition with a symmetrical behavior around a maximum value. After the thermal transition the aggregate was passed again on the same G-75 Sephadex column which has shown the peak relative to the native RNase, thus indicating a dissociation of the aggregate by heat. In fact, Crestfield (1) demonstrated that RNase aggregates, obtained by 50% acetic acid
BOVINE
PANCREATIC
375
RIBONUCLEASE
OD
i i i i i ;‘: __., i ; :,. i : ', I I L.,' i ::\. i
h.*.
200
EFFLUENT
400
(ml)
FIG. 2. Chromatographic behavior on a (1.8 X 140 cm) Sephadex G-75 column equilibrated and eluted with 0.1 M acetate buffer at pH 5 and 25”. Optical density (OD) measurements at 570 rnp, after calorimetric reaction with ninhydrin, are shown as a function of the elution volume. (a). (-) RNase A in acetate buffer pH 5, (---j RNase A + RNA at a ratio 1: 15.5 by weight, reacted for 5 min in 0.1 M acetate buffer pH 5, (- - - -) RNase A + RNA at the same ratio reacted for 6 hr, (-----) RNase A f RNA at the same ratio reacted for 24 hr; (b). (-.--) S-protein in acetate buffer pH 5, (-----) S-protein + RNA at a ratio 1:20 by weight reacted for 5 min in 0.1 M acetate buffer pH 5, (---) S-protein + RNA at the same ratio reacted for 24 hr in 0.1 M acetate buffer pH 5; (c). (+-) S-protein + S-peptide (in excess) + RNA at a ratio 1:20 by weight reacted for 24 hr in 0.1 M acetate pH 5; (d). (---) RNase A + cytidine-2’,3’-cyclic phosphate at a ratio 1: 12 by weight reacted at pH 7 and after 1 hr brought at pH 5. treatment, may be dissociated to monomers by heating at 65” It can be inferred, therefore, that the heating is able to dissociate any RNase aggregate. The phosphate analysis of the aggregation product yielded a value of 0.98 and 0.48%, respectively, for the product obtained using R.NA and cytidine as substrates. Assuming a molecular weight of about 370 for a single nucleotide, a fragment of polynucleotide equal to eight residues per protein molecule would be found to result using RNA as substrate and a 4-5-residues fragment would result if cytidine is used as a substrate. Activity
measurements
demonstrated
that
the RNase A aggregate still possesses 35-40% of the initial RNase activity measured as RNA hydrolysis. An aqueous solution (1 mg/ml) of the molecular aggregate containing 0.94 mg of protein and 0.06 mg of substrate, calculated on the basis of the phosphate content, shows a circular dichroism (CD) similar to that of native RNase both in absence and in presence of sodium dodecyl sulfate, but the dichroic bands are lower than the ones of native protein (Fig. 5). The CD bands for the two RNases (native and aggregate) have the same behavior with minima at 208 and 222 rnp and with a maximum at 194 rnp, but
376
MARZOTTO
ANI>
GALZIGNA
the magnitude of these bands is 40% lower for RNase aggregate than for native RNase A. The above values of wavelength for dichroic bands have been interpretedkrea
I
+6
t
t 00
I
I
I
,
I
/‘\ \
+4
E
/
i
+2
x 0
*s
0.7
06
0. 5
-8
0.4
-10
0. 3 I
190
02
210
200
/
220
230
240
t 01
L
I
230
250
270
290
310
330
.I
\
FIG. 3. Ultraviolet spectrum of an aqueous solution containing 0.5 mg/ml of RNase A molecular aggregate. The spectrum was carried out by means of a Perkin-Elmer spectrophotometer Model 165.
01’ 0
10
20
30
40
FIG. 5. Circular dichroism spect,rum in the far-ultraviolet of a 1 mg/ml aqueous solution of the molecular aggregate in the absence (+-) and in the presence (-----) of 0.05 M sodium dodecyl sulfate; [/I] was measured as a furlction of X (mH) by using a Roussel-Jouan (Paris) dichrograph. The dotted lines (-.-.-) and (- - -) represent the CD curves of native RNase A under the same conditions and, respectively, in the absence and in the presence of 0.05 M sodium dodecyl sulfate.
50
4. Thermal transition curve for a 1 mg/ml at 365 rnp is reported as a function of temperature. was employed. FIG.
250 1 CvJl
60
70
80
90 T’C
-
aqueous solution of the aggregate. A Perkin-Elmer polarimeter Model
[a] 141
BOVINE
PANCREATIC
RIBONUCLEASE
V/V” t 3.
2
2
1 I
3.2
3.4
3.6
3.8
4.0
42
4.4
4.6
4.6
5.0
fog M.W.--FIG. 6. Molecular weight determination by gel-filtratiou calibration curve. The molecular weights of RXase aggregates were estimated by the procedure of Whitaker (2): Sephadex G-75 column (140 X 1.8 cm) eluted with 0.1 nx sodium acetate pH 5; flow rate 0.5 ml;‘min; temperature 25”. The void volume (V,), was determined with blue dcxtran (Pharmacia, Uppsala), molecular weight 2. 106. Horse heart cytochrome c (type II), crystalline bovine serum albumin, and pepsin (twice crystallized) were obtained from Sigma, and crystalline sperm whale myoglobin was a produci of Iiock-Light Laboratories Ltd.
RSdue, respectively, to T-T* and II-K* transitions of peptide bond in a-helical conformat)ion (9, 10). Figure 6 reports the calibration curve for the gel-filtration methods with the elution volumes of the proteins used as standards. I’ and V,, are the elution volume and the void volume, respectively. The V/V, ratio is expressed as a function of the logarithm of the molecular weight (M.W.) of the proteins employed for such a calibration curve. The F/V0 ratio for RNse aggregates is shown on the curve. This aggregation phenomenon has been controlled by us both on different commercial samples of bovine ribonuclease and also on different preparation of horse pancreatic ribonuclease (unpublished data). The results obtained with different ribonucleases appear to be identical and independent from the type of ribonuclease used.
DISCUSSION
AND
CONCLUSION
The interaction between RNase A and substrates such as RNA and cytidine-%‘,3’cyclic phosphate brings about the formation of a molecular aggregate, whose concentration is increasing as a function of time. Such an aggregate between protein and substrate is formed also by S-protein alone and RNase S’ (E’igs. 2b, c). The molecular aggregation has a higher yield in the presence of the natural substrate, thus indicating an easier aggregation when the number of enzyme-substrate collisions is higher as should be the case of natural substrate. The uv spectrum and the activity measurements support the view that the aggregate is formed by the portion of the protein not involving the active site, bound to a fragment of substrate. This is also shown by the dissociation of protein from substrate
37s
MARZOTTO
AND
after heating and by the double and symmetrical transition temperature observed by thermal analysis. A diminished degree of ordering of the proteic part with the unblocked active site, which constitutes the molecular aggregate, appears from circular dichroism measurements in the far-ultraviolet which point to a lower content in the a-helix and in p-structure. The molecular weight of the molecular aggregates observed in the presence of both substrates corresponds to a value of approximately 29,000-30,000 for RNase A and RNase S’ and to a value of approximately 24,000 for S-protein. These findings suggest the necessity of taking into account such a phenomenon into the mechanism of RNase A-substrate interaction and the S-peptideS-protein recombination as well. Our results substantiate the hypothesis that dimerization is induced by the presence of substrate, the dimer being formed by two protein units
GALZIGNA
associated with a fragment of substrate having oligo-nucleotide dimensions. REFERENCES 1. CRESTFIELD, A.M., STEIN, W. H., ~NDMOORE, S., J. Biol. Chem. 238, 2421 (1963). 2. WHIT.~KER, J. R., dnd. Chem. 36, 1950 (1963). 3. HIRS, C. W., STEIN, W. H., .UD MOORE, S., J. Biol. Chem. 200, 493 (1953). 4. MARZOTTO, A, SCATTURIN, A., VIDNJ, G., -4~1) SCOFFONE, E., Gazz. Chim. Ital. 94, 760 (1964). 5. MARZOTTO, A., MARCHIORI, I?., MORODER, L., BONI, R., AND GALZIGNA, L., Biochim. Biophys. Acta 147, 26 (1967). 6. WELLNER, D., SILMAN, H. J., AND &LA, M., J. Biol. Chem. 238, 1324 (1963). 7. CARR, C. I., BND ZIMM, B. H., J. Chem. Phys. 18, 1616 (1950). 8. SHERWOOD, L. M., AND POTTS, J. T., J. Biol. Chem. 240, 3806 (1965). 9. TAMBURRO, A. M., SCATTURIN, A., .~ND MORODER, IL, Biochim. Biophys. dcta 164, 583 (1968). 10. HOLZWARTH, G., AND DOTY, P., J. Amer. Chem. Sot. 87, 218 (1965).
OF
BIOCHEWISTI~Y
Molecular
AND
137, 373-378 (1970)
BIOPHYSICS
Aggregation
of Bovine
Induced
cli Chimica
delle Macromolecole Macromoldcwlaire Received
Ribonuclease
by Substrate
A. 1\IARZOTTO’ Isktuto
Pancreatic
L. GALZIGNA2
AND
(C.N.R.), Padova, Italy and the Dkpartement (CRS) BP 1018 34 Montpellier, France
September
30, 1969; accepted
January
de Biochimie
8, 1970
The interaction between bovine cytidine-2’,3’-cyclic phosphate pancreatic ribonllclease and ribonucleic acid or cytidine-2’,3’-cyclic phosphate is studied by means of t,he gel-filtration method. The formation of a molecular aggregate of the enzyme as a function of time is shown. Some _ physicochemical properties of the aggregated prod\lct _ have been investigated.
It is well known (1) that specific experimental conditions can induce the formation of molecular aggregates of ribonuclease (RNase A) such as dimers, tetramers, and polymers. This study stems from our previous observation concerning the interaction with substrate which appeared to induce an aggregation of RNase A as well. We studied thoroughly such a phenomenon by means of gel filtration (2), by taking ribonuclease samples reacted with both natural (RNA) and synthetic substrate (cytidine-2’) 3’cyclic phosphate) at variable hydrolysis times. MATERIALS
AND
METHODS
RNase A was prepared according to t,he Hirs procedure (3), from commercial bovine ribonuclease (Serovac Laboratories). BPN’ bacterial protease (Nagarse) was purchased by Nagase Co. Ltd., Osaka, Japan. RNase S’ was prepared as described by Marzot,to (4) by proteolytic digestion of RNase A with Nagarse using a 2O:l weight ratio in a 0.1 M KC1 solution at 2-3”. The pH was maintained at 8 by addition of a 0.1 M NaOH solution. After the addition of 1.6 equivalents of NaOH per mole of protein, the reaction was stopped with standard FlCl. RNasr S was recovered by chromatography ’ Present, Address: Laboratorio Chimica Tecnologic Itadio-elementi C.N.B ., Padova, It,aly. 2 Contribution No. 38 from t,he Research Group (INSIj:RM)
on a CG 50 Amberlite column (5 X 60 cm) eluted with 0.2 M phosphat,e buffer at p1-I G.5. S-protein and S-peptide were separated with a G-100 Sephadex column eluted with 50(% acetic acid and then lyophilized. The depolymerase activity determination of RNaseI A RNase S’, and molecular aggregates were carrfied out as reported by Marzotto (5) [Ising RNA as a substrate. Yeast ribonllcleic acid (RNA) from British Drug Houses Ltd. purified before the use as indicated elsewhere (6) and cytidine-2’,3’-cyclic phosphate from Koch-Light Laboratories were used as substrates. The molecular weight of RNA rarlged between 140,000 and 160,000 as determined by the gel-filtration method of Whitaker (2) and by light-scattering measurements using a Sofica L.S. photometer Model. 42,000, with cylindrical cells immersed in highly purified toluene. The instrument was standardized with benzene (clarified with accuracy) (Ising Rw (4360 A) = 48.5 X 10-j (7). The measurements were made in 0.1 ~RI acetate buffer pH 5.7 and 0.2 M NaCl aqueous solution at, X = 4360 8 using the value dn/dc = 0.198. All the solutions used were clarified by prolouged shaking with chloroform-isoamyl alcohol mixture, followed by centrifugation at 25,000g for 2 hr. RNA concent,rations were determined by spectrophotometric measurements of the optical density at 257 m/l. The range of RNA concentrat,ions used was between 1.5 X 1OF and 1.5 X 1OF g/ml. The concentrations of RNase A, RNaseL S’ > and S-protein were determined by spectrophotometric measurements at 280 rnp (S), and the concentrations of the molecrdar ag373
374
MARZOTTO
AND
gregate were determined on the basis of the phosphate content. The circular dichroism (CD) measurements in the far-ultraviolet were performed by means of a Roussel-Jouan (Paris) dichrograph at 25”. Cylindrical opticell quartz cells were used with 0.05 cm optical path. The sensitivity was kept at 1 X 1W5. The samples contained 1 mg of the molecular aggregate per milliliter of an aqueous solution. The molecular weight were also calculated from a calibration curve obtained with pure samples of a number of standard proteins. Experiments of protein aggregation were carried out in the presence of RNA in 0.1 M acetate buffer pH 5 and cytidine-2’,3’ cyclic phosphate in buffer pH 7 at 25”. The weight 0.1 M Tris-HCl ratio between protein and substrate was 1:15-20 with RNA as a substrate as I:2 with cytidine as a substrate. In a typical experiment 0.090 g of RNase A dissolved in 3 ml of 0.1 M acetate buffer (pH 5) were added to 6 ml of acetate solution containing 1.4 g of RNA. Molecular aggregation was measured at different times by withdrawing from the reaction mixture 3-ml samples and by passing them through a G-75 Sephadex (1.8 X 140 cm) column equilibrated and eluted with 0.1 M acetate buffer at pH 5. A preliminary elution of the enzyme alone in absence of RNA was also done. The molecular weights were calculated by means of a calibrat,ion curve established with various proteins of different molecular weight. The aggregation product was desalted on a G-25 Sephadex column (1.8 X 140 cm) eluted with 5% acetic acid and then lyophylized. RESULTS
Figure 1 reports a saturation curve obtained at a fixed concentration of natural S-peptide and varying amounts of S-protein. Fractional activity (a) is expressed as a percent of the maximum value observed. The shape of this curve was tentatively ascribed to an aggregation effect and stimulated a check of the molecular weights of S-protein and RNase S’ in order to see if they could be influenced by the presence of the substrate. The chromatographic behavior on G-75 Sephadex of RNase A, S-protein, and RNase S’ before and after their interaction with substrate is presented in Fig. 2. The colorimetric reaction with ninhydrin was quantitated by optical density measurements at 570 rnfi.
GALZIGNA
::[ /-----TV06L
/
0.4
02
:
I 2
-
““’ 5
FIG. 1. Initial velocities of RNA depolymerization (a) expressed as a percent of the maximum value observed are reported as a function of the molar ratio (T) between increasing amounts of S-protein and a constant concentration (5.24.10-7 M) of S-peptide.
Figure 2a shows an evident increase of the peak corresponding to the molecular aggregate between RNase A and RNA and a corresponding decrease of the peak relative to native RNase as a function of the reaction time. The ultraviolet spectrum of an aqueous solution (0.5 mg/l ml) of the aggregated product corresponding to the chromatographic fraction with elution volume of 260 ml is given in Fig. 3, which shows a maximum value of absorbancy at 257 rnp, typical of RNA nucleotides, and a shoulder at 277 rnp, likely due to phenolic residues present in the protein. The thermal transition curve for the aqueous solution containing 1 mg/ml of the RNase molecular aggregate obtained as indicated in Fig. 2a is reported in Fig. 4. [01]at 365 rnb is given as a function of temperature. The curve shows a double transition with a symmetrical behavior around a maximum value. After the thermal transition the aggregate was passed again on the same G-75 Sephadex column which has shown the peak relative to the native RNase, thus indicating a dissociation of the aggregate by heat. In fact, Crestfield (1) demonstrated that RNase aggregates, obtained by 50% acetic acid
BOVINE
PANCREATIC
375
RIBONUCLEASE
OD
i i i i i ;‘: __., i ; :,. i : ', I I L.,' i ::\. i
h.*.
200
EFFLUENT
400
(ml)
FIG. 2. Chromatographic behavior on a (1.8 X 140 cm) Sephadex G-75 column equilibrated and eluted with 0.1 M acetate buffer at pH 5 and 25”. Optical density (OD) measurements at 570 rnp, after calorimetric reaction with ninhydrin, are shown as a function of the elution volume. (a). (-) RNase A in acetate buffer pH 5, (---j RNase A + RNA at a ratio 1: 15.5 by weight, reacted for 5 min in 0.1 M acetate buffer pH 5, (- - - -) RNase A + RNA at the same ratio reacted for 6 hr, (-----) RNase A f RNA at the same ratio reacted for 24 hr; (b). (-.--) S-protein in acetate buffer pH 5, (-----) S-protein + RNA at a ratio 1:20 by weight reacted for 5 min in 0.1 M acetate buffer pH 5, (---) S-protein + RNA at the same ratio reacted for 24 hr in 0.1 M acetate buffer pH 5; (c). (+-) S-protein + S-peptide (in excess) + RNA at a ratio 1:20 by weight reacted for 24 hr in 0.1 M acetate pH 5; (d). (---) RNase A + cytidine-2’,3’-cyclic phosphate at a ratio 1: 12 by weight reacted at pH 7 and after 1 hr brought at pH 5. treatment, may be dissociated to monomers by heating at 65” It can be inferred, therefore, that the heating is able to dissociate any RNase aggregate. The phosphate analysis of the aggregation product yielded a value of 0.98 and 0.48%, respectively, for the product obtained using R.NA and cytidine as substrates. Assuming a molecular weight of about 370 for a single nucleotide, a fragment of polynucleotide equal to eight residues per protein molecule would be found to result using RNA as substrate and a 4-5-residues fragment would result if cytidine is used as a substrate. Activity
measurements
demonstrated
that
the RNase A aggregate still possesses 35-40% of the initial RNase activity measured as RNA hydrolysis. An aqueous solution (1 mg/ml) of the molecular aggregate containing 0.94 mg of protein and 0.06 mg of substrate, calculated on the basis of the phosphate content, shows a circular dichroism (CD) similar to that of native RNase both in absence and in presence of sodium dodecyl sulfate, but the dichroic bands are lower than the ones of native protein (Fig. 5). The CD bands for the two RNases (native and aggregate) have the same behavior with minima at 208 and 222 rnp and with a maximum at 194 rnp, but
376
MARZOTTO
ANI>
GALZIGNA
the magnitude of these bands is 40% lower for RNase aggregate than for native RNase A. The above values of wavelength for dichroic bands have been interpretedkrea
I
+6
t
t 00
I
I
I
,
I
/‘\ \
+4
E
/
i
+2
x 0
*s
0.7
06
0. 5
-8
0.4
-10
0. 3 I
190
02
210
200
/
220
230
240
t 01
L
I
230
250
270
290
310
330
.I
\
FIG. 3. Ultraviolet spectrum of an aqueous solution containing 0.5 mg/ml of RNase A molecular aggregate. The spectrum was carried out by means of a Perkin-Elmer spectrophotometer Model 165.
01’ 0
10
20
30
40
FIG. 5. Circular dichroism spect,rum in the far-ultraviolet of a 1 mg/ml aqueous solution of the molecular aggregate in the absence (+-) and in the presence (-----) of 0.05 M sodium dodecyl sulfate; [/I] was measured as a furlction of X (mH) by using a Roussel-Jouan (Paris) dichrograph. The dotted lines (-.-.-) and (- - -) represent the CD curves of native RNase A under the same conditions and, respectively, in the absence and in the presence of 0.05 M sodium dodecyl sulfate.
50
4. Thermal transition curve for a 1 mg/ml at 365 rnp is reported as a function of temperature. was employed. FIG.
250 1 CvJl
60
70
80
90 T’C
-
aqueous solution of the aggregate. A Perkin-Elmer polarimeter Model
[a] 141
BOVINE
PANCREATIC
RIBONUCLEASE
V/V” t 3.
2
2
1 I
3.2
3.4
3.6
3.8
4.0
42
4.4
4.6
4.6
5.0
fog M.W.--FIG. 6. Molecular weight determination by gel-filtratiou calibration curve. The molecular weights of RXase aggregates were estimated by the procedure of Whitaker (2): Sephadex G-75 column (140 X 1.8 cm) eluted with 0.1 nx sodium acetate pH 5; flow rate 0.5 ml;‘min; temperature 25”. The void volume (V,), was determined with blue dcxtran (Pharmacia, Uppsala), molecular weight 2. 106. Horse heart cytochrome c (type II), crystalline bovine serum albumin, and pepsin (twice crystallized) were obtained from Sigma, and crystalline sperm whale myoglobin was a produci of Iiock-Light Laboratories Ltd.
RSdue, respectively, to T-T* and II-K* transitions of peptide bond in a-helical conformat)ion (9, 10). Figure 6 reports the calibration curve for the gel-filtration methods with the elution volumes of the proteins used as standards. I’ and V,, are the elution volume and the void volume, respectively. The V/V, ratio is expressed as a function of the logarithm of the molecular weight (M.W.) of the proteins employed for such a calibration curve. The F/V0 ratio for RNse aggregates is shown on the curve. This aggregation phenomenon has been controlled by us both on different commercial samples of bovine ribonuclease and also on different preparation of horse pancreatic ribonuclease (unpublished data). The results obtained with different ribonucleases appear to be identical and independent from the type of ribonuclease used.
DISCUSSION
AND
CONCLUSION
The interaction between RNase A and substrates such as RNA and cytidine-%‘,3’cyclic phosphate brings about the formation of a molecular aggregate, whose concentration is increasing as a function of time. Such an aggregate between protein and substrate is formed also by S-protein alone and RNase S’ (E’igs. 2b, c). The molecular aggregation has a higher yield in the presence of the natural substrate, thus indicating an easier aggregation when the number of enzyme-substrate collisions is higher as should be the case of natural substrate. The uv spectrum and the activity measurements support the view that the aggregate is formed by the portion of the protein not involving the active site, bound to a fragment of substrate. This is also shown by the dissociation of protein from substrate
37s
MARZOTTO
AND
after heating and by the double and symmetrical transition temperature observed by thermal analysis. A diminished degree of ordering of the proteic part with the unblocked active site, which constitutes the molecular aggregate, appears from circular dichroism measurements in the far-ultraviolet which point to a lower content in the a-helix and in p-structure. The molecular weight of the molecular aggregates observed in the presence of both substrates corresponds to a value of approximately 29,000-30,000 for RNase A and RNase S’ and to a value of approximately 24,000 for S-protein. These findings suggest the necessity of taking into account such a phenomenon into the mechanism of RNase A-substrate interaction and the S-peptideS-protein recombination as well. Our results substantiate the hypothesis that dimerization is induced by the presence of substrate, the dimer being formed by two protein units
GALZIGNA
associated with a fragment of substrate having oligo-nucleotide dimensions. REFERENCES 1. CRESTFIELD, A.M., STEIN, W. H., ~NDMOORE, S., J. Biol. Chem. 238, 2421 (1963). 2. WHIT.~KER, J. R., dnd. Chem. 36, 1950 (1963). 3. HIRS, C. W., STEIN, W. H., .UD MOORE, S., J. Biol. Chem. 200, 493 (1953). 4. MARZOTTO, A, SCATTURIN, A., VIDNJ, G., -4~1) SCOFFONE, E., Gazz. Chim. Ital. 94, 760 (1964). 5. MARZOTTO, A., MARCHIORI, I?., MORODER, L., BONI, R., AND GALZIGNA, L., Biochim. Biophys. Acta 147, 26 (1967). 6. WELLNER, D., SILMAN, H. J., AND &LA, M., J. Biol. Chem. 238, 1324 (1963). 7. CARR, C. I., BND ZIMM, B. H., J. Chem. Phys. 18, 1616 (1950). 8. SHERWOOD, L. M., AND POTTS, J. T., J. Biol. Chem. 240, 3806 (1965). 9. TAMBURRO, A. M., SCATTURIN, A., .~ND MORODER, IL, Biochim. Biophys. dcta 164, 583 (1968). 10. HOLZWARTH, G., AND DOTY, P., J. Amer. Chem. Sot. 87, 218 (1965).