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Adsorption of ribonucleic acid on bentonite
ANALYTICAL
BIOCHEMISTRY
32,
15o-lfi‘i (1969)
SHORT COMMUNICATION
Adsorption
of
Ribonucleic
Acid
on
Bentonitel
Since the original observation by Brownhill et al. (1) that bentonite was an inhibitor of nucleases, bentonite, and other diatomaceous earths have been routinely used in the preparation of ribosomes and RNA from cells. One of the first procedures for the preparation of RNA using bentonite was by Fraenkel-Conrat, Singer, and Tsugita (2) using tobacco mosaic virus. They observed that bentonite, through the protection of RNA by binding of ribonuclease, enhanced the reconstitution of active virus particles from TMV protein and TMV-RNA (3). Fraenkel-Conrat (4) later reported that bentonite would adsorb some RNA. Recently, Hsiao (5) reported the use of bentonite in the inhibition of RNAase associated with the ribosomes of Zea muys and also reported an effect of bentonite on ribosomal yield. At a level of 2 mg/ml, bentonite decreased the yield of ribosomes by 95%. Tester and Dure (6) had earlier reported a selective precipitation of monomeric ribosomes from oatseeds by bentonite. However, Hsiao reported that ribosomal RNA was not adsorbed onto bentonite under his experimental conditions. These observations were attributed to a simple electrostatic effect of the negatively charged bentonite binding the positively charged ribonuclease, binding with the positively charged protein portion of the ribosome and repelling the negatively charged ribosomal-RNA. However, the binding of polyelectrolytes to charged surfaces involves forces other than electrostatic interactions. Winsten (7) has proposed that van der Waals forces as well as hydrophobic bond formation may be important in the binding of polyribonucleotides. Bull (8) observed the binding of positively charged bovine serum albumin to Dowex 2 anion-exchange resin and of negatively charged bovine serum albumin to Dowex 50 cation-exchange resin. Chattoraj and co-workers (9, 10) have observed the binding of both DNA and RNA to negatively charged Dowex 50 resin particles. ‘This work was supported Health and by Hatch Project
by grant 86190.
GM-11672 150
of
the
National
Institutes
of
SHORT
151
COMMUNICATION
The purpose of this investigation is to study the effect of ribosomalRNA on bentonite in order to see if binding does occur and, if so, to measure the extent of this binding. Methods. Ribosomal-RNA was prepared from Escherichiu co& strain K,, (HFr) W3780, by the method of Stanley (11). Lower molecular weight RNA’s were removed by precipitation of the ribosomal-RNA from the aqueous phase of the phenolic extract with 2 M NaCl. Bentonite was washed and sized by the procedure of Fraenkel-Conrat et al. (2) before
I
-20 0
0.02
004
006 (%
I 008
I --.1-- !
0 IO
012
Fmn,+
FIG. 1. Electrophoretic mob&ties of bentonite particles as function of square root of per cent ribosomal-RNA concentration (w/v) at ionic strength of 0.1 in sodium acetate buffer at pH 6.1 and at 25”. The dashed line is mobility of yeastRNA and Dowex 50 in NaCl at an ionic strength of 0.05 and a pH of 6.5 and 30” (10).
use. Dowex 50-X12 resin was exchanged against KOH and HCl and KOH and then thoroughly washed before use. Both particles were about 4 p in size. Concentrations of all preparations and suspensionswere determined by drying in a vacuum oven at 105”. All other chemicals were reagent grade and all solutions were made with quartz-distilled water. Microelectrophoretic measurements were conducted using a Zeiss Cytopherometer in a horizontally oriented cell which was thermostated at 25”. Adsorption studies were performed by incubating, for 30 min at 25”, 2.5 mg of ribosomal-RNA and 2 mg of a suspensionof either ben-
152
SHORT
COMMUNICATION
tonite or Dowex 50 particles in a total volume of 20 ml, which contained sodium acetate buffer at a pH of 6.1 and an ionic strength of 0.1. The particles were collected on a Millipore (0.45 F), washed with buffer, and assayed for RNA-phosphate by the method of Fiske and SubbaRow (12) using ribosomal-RNA as a standard. Results and Discussion. In Figure 1 are presented the data for the microelectrophoretic mobilities of bentonite particles as a function of the square root of the concentration of ribosomal-RNA in sodium acetate buffer at pH 6.1 and at an ionic strength of 0.1. Upon the addition of ribosomal-RNA, the mobility of bentonite particles changed from a value of -3.1 ,p/sec/v/cm to a constant value of -2.6 p/set/v/cm above a ribosomal-RNA concentration of 0.005% (w/v). Chowrashi et al. (10) had observed that, when yeast-RNA was added to Dowex 50 resin in NaCl-KOH at an ionic strength of 0.05 and at a pH of 6.5, the mobilities of the Dowex 50 resin particles changed from -2.4 p/set/v/cm to -3.0 CL/ set/v/cm at a RNA concentration above 0.005%. Thus, ribosomal-RNA does seem to bind to the surface of bentonite and cause a change in the electrophoretic mobility of these particles and the concentrations of ribosomal-RNA at which saturation of the be&mite surface occurs is at the same level as was observed with the Dowex 50 yeast-RNA system. However, the mobility of ribosomal-RNA coated bentonite (-2.6 &sec/ v/cm) is somewhat lower than that of yeast-RNA coated Dowex 50 (-3.0 &ec/v/cm). Since the underlying surface has been observed to have little effect on the microelectrophoretic mobility of adsorbed biopolymers (8, 13)) this difference in mobility might reflect structural differences between these RNA%. Olivera et al. (14) reported a moving boundary electrophoretic mobility of -1.7 &sec/v/cm for ribosomalRNA from animal cells. The amounts of ribosomal-RNA adsorbed on bentonite and on Dowex 50 resin are reported in Table 1. It should be mentioned here that several other methods were attempted to measure the amount of RNA adsorbed, onto these particles. These include first measuring the decrease in 260 nm
Adsorption
of Ribosomal-RN
TABLE A onto
1 Bentonite
and Dowex
5OW-X12
pg ribosomal-RNA Particle Dowex 5OW-X12 Bentonite
mg particle 17 50
SHORT
COMMUNICATION
153
adsorbing material after removing the particles by centrifugation or by filtration (which failed because immeasurable RNA was removed) and, second, determining by the orcinol method the ribose content which was associated with the particles after adsorption. This failed because extremely high blanks were obtained from the resin particles when they were treated with t,he orcinol reagent. Since both particles were sized to yield particles about 4 J” large, 1 gm of particles provided a surface area of about 1.5 meter2, assuming that the particles were spherical with a density of 1. Therefore, the amount of adsorption of ribosomal-RNA to bentonite was 33 mg/meter? or 50 pg/mg, whereas the values on Dowex 50 were 12 mg/mete? or 17 pg/mg. Bull (1516) observed that the amount of protein that was tightly bound to glass was about 1 mg/meteP. For comparison purposes, the value of RNA concentration in Figure 1 at which saturation of the surface occurred was 2 mg RNA/mg bentonite. Hsiao reported no adsorption of ribosomal-RNA at a ratio of 2 mg of RNA/mg bentonite. The data reported in this paper show that bentonite will adsorb appreciable amounts of E. coli ribosomal-RNA and that Fraenkel-Conrat’s (4) warning about the indiscriminate use of bentonite in the preparation of RNA samples should be heeded. Hsiao’s (5) observation that there was no adsorption of Zea mays ribosomal-RNA onto bentonite at ratios of 2 mg of RNA/mg bentonite has not been supported. No mention was made by him as to the method used to measure binding. REFERENCES 1. BROWNHILL, T. J., JONES, A. S., .~ND STACEY, M., B&hem. J. 73, 434 (1959). 2. FRAENKEL-CONRAT, H., SINGER, B., AND TSUGIT~, A., Virology 14, 54 (1961). 3. SINGER, B., AND FRAENKEL-CONRAT, H., v&logy 14, 59 (1961). 4. FRAENKEL-CONRAT, H., in “Procedures in Nucleic Acid Research” (G. L. Gantoni and D. R. Davies, eds.), p. 435. Harper & Row, New York, 1966. 5. HSIAO, T. C., Plant Physiol. 43, 1355 (1968). 6. TESTER, C. F., .~ND DURE, L., Biochem. Biophys. Res. Commun. 23, 287 (1966). 7. WINSTEN, W. A., Biopolymers 2, 337 (1964). 8. BULL, H. B., Arch. Biochem. Biophys. 98, 427 (1962). 9. CHATTOI&~J. D. Ii., CHOWRASHI, P. Ii., .~ND ~H.IECR.~VARTI, K., Biopolymers 5, 173 (1967). 10. CHOWRASHI, P. K., CHATTORAJ, D. Ii., ANI) CHAKRAVARTI, K., Biopolymers 6, 97 (1968). 11. STANLEY, W. M.. JR., “Physical Studies on the Ribosomes and Ribosomal Ribonucleic Acid of Escherichia coli," Ph.D. Dissertation, University of Wisconsin, 1963. 12. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 81, 629 (1929). 13. MILLER, I. R., Biochim. Biophys. Acta 103, 219 (1965). 14. OLIVERA, B. M., BAINE, P., AND D.~VIDSON, N., Biopolymers 2, 245 (1964). 15. BULL, H. B., Arch. Biochem. Biophys. 68, 102 (1957). 16. BULL, H. B., Biochim. Biophys. Acta 19, 464 (1956).
154
SHORT COMMUNICATIONS
M. V. BLANTON' L. B. BARNETT Department
of Biochemistry Polytechnic Instituite Blacksburg, Virginia 24061 Received May 1.2, 1969
and
Nutrition
Virginia
’ Present
address:
Detection
Anheuser-Busch,
of
Corn
Products
Proteolytic Enzymes Acetate Electrophoresis
Division,
after
St. Louis,
Missouri.
Cellulose
Uriel (1) developed a method for detection of proteolytic enzymes after electrophoresis in agar gel. After completion of electrophoresis the agar plate is immersed in a solution of a susceptible protein such as casein. After incubation for an appropriate period of time, the plate is fixed, dried, and stained for protein. Areas of proteolytic activity are detected by dimunition or absence of color. Merkel (2) more recently suggested a procedure applicable to cellulose acetate electrophoresis, whereby proteolysis decolorizes an algal chromoprotein in an agar matrix on which the strip is applied. The agar gel then is scanned visually or densitometrically. A method whereby the pattern of proteolytic activity of separated bands is detected in the strip itself was developed in the course of a study of the extracellular proteinase of Streptococcus faeculis var. liquefaciens. It has been used more recently with other proteolytic preparations. The conditions described here were found satisfactory for the enzymes studied by us. Slight modifications may be necessary for other proteolytic enzymes. Enzymes. The proteinase of S. faecalis var. liquefaciens was prepared within dialysis tubing in the apparatus described elsewhere (3). Bacterial cells were removed from the dialysis retentate by centrifugation, and the concentrated enzyme in the supernate was used for electrophoresis. Pancreatin (Pangestin 1:75, lot 440641) was obtained from Difco Laboratories, Detroit, Michigan. Trypsin (salt-free 2 X crystalline, lot 9680) was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio.
BIOCHEMISTRY
32,
15o-lfi‘i (1969)
SHORT COMMUNICATION
Adsorption
of
Ribonucleic
Acid
on
Bentonitel
Since the original observation by Brownhill et al. (1) that bentonite was an inhibitor of nucleases, bentonite, and other diatomaceous earths have been routinely used in the preparation of ribosomes and RNA from cells. One of the first procedures for the preparation of RNA using bentonite was by Fraenkel-Conrat, Singer, and Tsugita (2) using tobacco mosaic virus. They observed that bentonite, through the protection of RNA by binding of ribonuclease, enhanced the reconstitution of active virus particles from TMV protein and TMV-RNA (3). Fraenkel-Conrat (4) later reported that bentonite would adsorb some RNA. Recently, Hsiao (5) reported the use of bentonite in the inhibition of RNAase associated with the ribosomes of Zea muys and also reported an effect of bentonite on ribosomal yield. At a level of 2 mg/ml, bentonite decreased the yield of ribosomes by 95%. Tester and Dure (6) had earlier reported a selective precipitation of monomeric ribosomes from oatseeds by bentonite. However, Hsiao reported that ribosomal RNA was not adsorbed onto bentonite under his experimental conditions. These observations were attributed to a simple electrostatic effect of the negatively charged bentonite binding the positively charged ribonuclease, binding with the positively charged protein portion of the ribosome and repelling the negatively charged ribosomal-RNA. However, the binding of polyelectrolytes to charged surfaces involves forces other than electrostatic interactions. Winsten (7) has proposed that van der Waals forces as well as hydrophobic bond formation may be important in the binding of polyribonucleotides. Bull (8) observed the binding of positively charged bovine serum albumin to Dowex 2 anion-exchange resin and of negatively charged bovine serum albumin to Dowex 50 cation-exchange resin. Chattoraj and co-workers (9, 10) have observed the binding of both DNA and RNA to negatively charged Dowex 50 resin particles. ‘This work was supported Health and by Hatch Project
by grant 86190.
GM-11672 150
of
the
National
Institutes
of
SHORT
151
COMMUNICATION
The purpose of this investigation is to study the effect of ribosomalRNA on bentonite in order to see if binding does occur and, if so, to measure the extent of this binding. Methods. Ribosomal-RNA was prepared from Escherichiu co& strain K,, (HFr) W3780, by the method of Stanley (11). Lower molecular weight RNA’s were removed by precipitation of the ribosomal-RNA from the aqueous phase of the phenolic extract with 2 M NaCl. Bentonite was washed and sized by the procedure of Fraenkel-Conrat et al. (2) before
I
-20 0
0.02
004
006 (%
I 008
I --.1-- !
0 IO
012
Fmn,+
FIG. 1. Electrophoretic mob&ties of bentonite particles as function of square root of per cent ribosomal-RNA concentration (w/v) at ionic strength of 0.1 in sodium acetate buffer at pH 6.1 and at 25”. The dashed line is mobility of yeastRNA and Dowex 50 in NaCl at an ionic strength of 0.05 and a pH of 6.5 and 30” (10).
use. Dowex 50-X12 resin was exchanged against KOH and HCl and KOH and then thoroughly washed before use. Both particles were about 4 p in size. Concentrations of all preparations and suspensionswere determined by drying in a vacuum oven at 105”. All other chemicals were reagent grade and all solutions were made with quartz-distilled water. Microelectrophoretic measurements were conducted using a Zeiss Cytopherometer in a horizontally oriented cell which was thermostated at 25”. Adsorption studies were performed by incubating, for 30 min at 25”, 2.5 mg of ribosomal-RNA and 2 mg of a suspensionof either ben-
152
SHORT
COMMUNICATION
tonite or Dowex 50 particles in a total volume of 20 ml, which contained sodium acetate buffer at a pH of 6.1 and an ionic strength of 0.1. The particles were collected on a Millipore (0.45 F), washed with buffer, and assayed for RNA-phosphate by the method of Fiske and SubbaRow (12) using ribosomal-RNA as a standard. Results and Discussion. In Figure 1 are presented the data for the microelectrophoretic mobilities of bentonite particles as a function of the square root of the concentration of ribosomal-RNA in sodium acetate buffer at pH 6.1 and at an ionic strength of 0.1. Upon the addition of ribosomal-RNA, the mobility of bentonite particles changed from a value of -3.1 ,p/sec/v/cm to a constant value of -2.6 p/set/v/cm above a ribosomal-RNA concentration of 0.005% (w/v). Chowrashi et al. (10) had observed that, when yeast-RNA was added to Dowex 50 resin in NaCl-KOH at an ionic strength of 0.05 and at a pH of 6.5, the mobilities of the Dowex 50 resin particles changed from -2.4 p/set/v/cm to -3.0 CL/ set/v/cm at a RNA concentration above 0.005%. Thus, ribosomal-RNA does seem to bind to the surface of bentonite and cause a change in the electrophoretic mobility of these particles and the concentrations of ribosomal-RNA at which saturation of the be&mite surface occurs is at the same level as was observed with the Dowex 50 yeast-RNA system. However, the mobility of ribosomal-RNA coated bentonite (-2.6 &sec/ v/cm) is somewhat lower than that of yeast-RNA coated Dowex 50 (-3.0 &ec/v/cm). Since the underlying surface has been observed to have little effect on the microelectrophoretic mobility of adsorbed biopolymers (8, 13)) this difference in mobility might reflect structural differences between these RNA%. Olivera et al. (14) reported a moving boundary electrophoretic mobility of -1.7 &sec/v/cm for ribosomalRNA from animal cells. The amounts of ribosomal-RNA adsorbed on bentonite and on Dowex 50 resin are reported in Table 1. It should be mentioned here that several other methods were attempted to measure the amount of RNA adsorbed, onto these particles. These include first measuring the decrease in 260 nm
Adsorption
of Ribosomal-RN
TABLE A onto
1 Bentonite
and Dowex
5OW-X12
pg ribosomal-RNA Particle Dowex 5OW-X12 Bentonite
mg particle 17 50
SHORT
COMMUNICATION
153
adsorbing material after removing the particles by centrifugation or by filtration (which failed because immeasurable RNA was removed) and, second, determining by the orcinol method the ribose content which was associated with the particles after adsorption. This failed because extremely high blanks were obtained from the resin particles when they were treated with t,he orcinol reagent. Since both particles were sized to yield particles about 4 J” large, 1 gm of particles provided a surface area of about 1.5 meter2, assuming that the particles were spherical with a density of 1. Therefore, the amount of adsorption of ribosomal-RNA to bentonite was 33 mg/meter? or 50 pg/mg, whereas the values on Dowex 50 were 12 mg/mete? or 17 pg/mg. Bull (1516) observed that the amount of protein that was tightly bound to glass was about 1 mg/meteP. For comparison purposes, the value of RNA concentration in Figure 1 at which saturation of the surface occurred was 2 mg RNA/mg bentonite. Hsiao reported no adsorption of ribosomal-RNA at a ratio of 2 mg of RNA/mg bentonite. The data reported in this paper show that bentonite will adsorb appreciable amounts of E. coli ribosomal-RNA and that Fraenkel-Conrat’s (4) warning about the indiscriminate use of bentonite in the preparation of RNA samples should be heeded. Hsiao’s (5) observation that there was no adsorption of Zea mays ribosomal-RNA onto bentonite at ratios of 2 mg of RNA/mg bentonite has not been supported. No mention was made by him as to the method used to measure binding. REFERENCES 1. BROWNHILL, T. J., JONES, A. S., .~ND STACEY, M., B&hem. J. 73, 434 (1959). 2. FRAENKEL-CONRAT, H., SINGER, B., AND TSUGIT~, A., Virology 14, 54 (1961). 3. SINGER, B., AND FRAENKEL-CONRAT, H., v&logy 14, 59 (1961). 4. FRAENKEL-CONRAT, H., in “Procedures in Nucleic Acid Research” (G. L. Gantoni and D. R. Davies, eds.), p. 435. Harper & Row, New York, 1966. 5. HSIAO, T. C., Plant Physiol. 43, 1355 (1968). 6. TESTER, C. F., .~ND DURE, L., Biochem. Biophys. Res. Commun. 23, 287 (1966). 7. WINSTEN, W. A., Biopolymers 2, 337 (1964). 8. BULL, H. B., Arch. Biochem. Biophys. 98, 427 (1962). 9. CHATTOI&~J. D. Ii., CHOWRASHI, P. Ii., .~ND ~H.IECR.~VARTI, K., Biopolymers 5, 173 (1967). 10. CHOWRASHI, P. K., CHATTORAJ, D. Ii., ANI) CHAKRAVARTI, K., Biopolymers 6, 97 (1968). 11. STANLEY, W. M.. JR., “Physical Studies on the Ribosomes and Ribosomal Ribonucleic Acid of Escherichia coli," Ph.D. Dissertation, University of Wisconsin, 1963. 12. FISKE, C. H., AND SUBBAROW, Y., J. Biol. Chem. 81, 629 (1929). 13. MILLER, I. R., Biochim. Biophys. Acta 103, 219 (1965). 14. OLIVERA, B. M., BAINE, P., AND D.~VIDSON, N., Biopolymers 2, 245 (1964). 15. BULL, H. B., Arch. Biochem. Biophys. 68, 102 (1957). 16. BULL, H. B., Biochim. Biophys. Acta 19, 464 (1956).
154
SHORT COMMUNICATIONS
M. V. BLANTON' L. B. BARNETT Department
of Biochemistry Polytechnic Instituite Blacksburg, Virginia 24061 Received May 1.2, 1969
and
Nutrition
Virginia
’ Present
address:
Detection
Anheuser-Busch,
of
Corn
Products
Proteolytic Enzymes Acetate Electrophoresis
Division,
after
St. Louis,
Missouri.
Cellulose
Uriel (1) developed a method for detection of proteolytic enzymes after electrophoresis in agar gel. After completion of electrophoresis the agar plate is immersed in a solution of a susceptible protein such as casein. After incubation for an appropriate period of time, the plate is fixed, dried, and stained for protein. Areas of proteolytic activity are detected by dimunition or absence of color. Merkel (2) more recently suggested a procedure applicable to cellulose acetate electrophoresis, whereby proteolysis decolorizes an algal chromoprotein in an agar matrix on which the strip is applied. The agar gel then is scanned visually or densitometrically. A method whereby the pattern of proteolytic activity of separated bands is detected in the strip itself was developed in the course of a study of the extracellular proteinase of Streptococcus faeculis var. liquefaciens. It has been used more recently with other proteolytic preparations. The conditions described here were found satisfactory for the enzymes studied by us. Slight modifications may be necessary for other proteolytic enzymes. Enzymes. The proteinase of S. faecalis var. liquefaciens was prepared within dialysis tubing in the apparatus described elsewhere (3). Bacterial cells were removed from the dialysis retentate by centrifugation, and the concentrated enzyme in the supernate was used for electrophoresis. Pancreatin (Pangestin 1:75, lot 440641) was obtained from Difco Laboratories, Detroit, Michigan. Trypsin (salt-free 2 X crystalline, lot 9680) was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio.