Resolution of Ribonucleotides by Zone Electrophoresis ARTHUR M. CRESTFIELD and FRANK WORTHINGTON ALLEN Department of Physiological Chemistry, University of California School of Medicine, Berkeley, Calif.
strength. This resolution has also been found by other workers (9, 16, 1 8 , f 9 ) . Separation of the four 3' mononucleotides occurs also in 90 minutes with a field of 30 volts per em. a t a pH of 9.2 in 0.1M sodium tetraborate buffer, as shown in Figure 1,B. This resolution is expected from the presence of enolic group dissociations in this pH region in uridylic and guanylic acids, the higher pK for the enol group of uridylic acid than for that of guanylic acid, as well as the lower mass of the uridylic acid, and the difference in mass between the cytidylic and adenylic acids. Mixtures of the mononucleotides may he fractionated best according t o the base component by the conditions at pH 3.5, as the percentage difference in mobilities is higher than a t pH 9.2. Separation of Each 5' Isomer from Corresponding 2' and 3' Isomers. The 5' phosphate ester of any ribose nucleoside possesses one characteristic by means of which it may be separated from the 2' and 3' esters of this same nucleoside: the presence of two adjacent hydroxyl groups in the ribose moiety. Complexcs of boric acid with adjacent hydroxyl groups have been reviewed recently by Boeseken (2) and Zittle ( 2 0 ) . The reaction generally is reported to occur in sodium tetraborate solution a t pH 9.2. The complex is a stronger acid than boric acid itself (&7), and hence it is strongly negative a t pH 9.2 (20). The mobility of the 5' nucleotide in tetraborate buffer is expected to
The study reports the electrophoretic separation of the isomeric ribonucleotides by conditions which emphasize one or more of the following properties of the molecules: the net charge, the mass and geometry, and the formation of complexes. With the exceptions of the 2' and 3' isomers of guanylic acid and the 3'and 5' isomers of adenylic acid, the resolutions described have not been previously obtained by zone electrophoretic techniques. One advantage of the new method is the speed with which the separations may be achieved.
C
HROMATOGRAPHIC and spectrophotometric procedures for the analysis of the mononucleotide composition of ribonucleic acids have received widespread attention since the initial work of Vischer and Chargaff (f7). Effort has been expended upon conditions for the liberation of the various mononucleotides, their separation, and their quantitative determination. No one of the paper chromatographic solvents described in the literature has been reported to resolve the mononucleotides completely and subsequently permit quantitative estimation by ultraviolet spectrophotometry ( S , 4 , 10,16). At present three isomers of each of the four ribonucleotides are recognized. These isomers have been characterized as the 2', 3', or 5' phosphate esters of adenosine, guanosine, cytidine, or uridine. Their presence in a hydrolyzate of ribonucleic acids is dependent upon the hydrolytic conditions chosen. Separation is accomplished by ion exchange chromatography (6). The present study reports the electrophoretic separation of the isomeric nucleotides by conditions which emphasize one or more of the following properties of the molecules: the net charge, the mass and geometry, and the formation of complexes.
di
A3
53
"3
0 00 0
B
lI DC 1/
METHOD
The apparatus and techniques have been described (8). The best conditions for a given resolution by electrophoresis have been selected by consideration of the net charges of the nucleotide molecules as calculated from published dissociation constants, inclusion of mass differences among the nucleotides as frictional differences, calculation of net charge and mass upon formation of complexes with boric acid, experimental verification of the resolutions under the chosen conditions, and incidental observation of unexpected resolutions during experiments which a-ere designed for other purposes. Before it is concluded that a resolution of two substances is possible, certain precautions must be observed: The mobilities must be compared in one and the same sheet of paper, and a mixture of the two substances must be included in a third channel of the paper.
L
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0
c3
c5
I
I I
0 0 G3
0 0
00
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I
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Figure 1. Resolutions of nucleotides A , Q, C, and U refer t o adenylic, guanylic,, cytidylic, and uridylic acids,
RESULTS AND DISCUSSION
respectively. Superscript refers to appropriate isomer. shown is 28 cm.
Separation of 3' Mononucleotides. The titration curves of the four ribonucleotides have been determined by Levene and coworkers ( l a , IS). These data are for the 3' isomers (6). It is evident from these curves that within the region of pH 3 to 4, the net charges on the nucleotides are sufficiently different t o make possible complete separation in an electrical field due to the differences in the dissociation constants of the amino groups and the absence of an amino group in uridylic acid. Figure 1,A, shows the resolution which is obtained in 60 minutes with a field of 30 volts per cm. at pH 3.5 in formate buffer of 0.1 ionic
A.
Length of paper
Formate buffer, p H 3.5, ionic stfength 0.1, field 30 volts per om., time 60 minutes
B . 0.1M sodium tetraborate solutiqn, p H 9.2, field 30 volts per cm., time 93
minutes 0.1M sodium tetraborate solution, p H 9.2, field 37 volts per cm., time 90 minutee Q. 0.1M sodium bicarbonate, pH.8.0, field 40 volts per om., time 120 minutes H. Formate buffer, p H 3.8, ionic strength 0.1,field 38 volts per cm., time 240 minutes I. Phosphate buffer, p H 6.2, ionic strength 0.1, field 32 volts per om., time 125 minutes J . Phosphate bnffer, p H 5.8, ionic strength 0.1, field 37 volts per em., time 148 minutes
C-F.
424
V O L U M E 27, N O . 3, M A R C H 1 9 5 5
425
be faster than that of the corresponding 2‘ and 3’ nucleotides. Such resolutions were obtained in 90 minutes with a field of 3T volts per em. a t p H 9.2 in 0.lM sodium tetraborate buffer, as shown in Figure l , C to F . Similar results for t h e adenylic acid isomers were obtained by Jaenicke and Vollbrechtshausen (11). Separation of 2’ Isomer from Corresponding 3’ Isomer. The dissociation constants of the 2‘ and 3’ isomers of adenylic acid and cytidylic acid differ appreciably ( 1 , 5 ) . Resolution of these isomers is expected to occur near the DH values where dissociations are half completed ( 7 ) . Experiments showed that the 2‘ and 3’ isomers of both pyrimidine nucleotides could be resolved in the region of p H 6 in 0.1 ionic strength phosphate buffer Figure 1,1and J . I t is likely that the separations occur because of net charge differences, as the cytidylic 3‘ isomer moves faster than thc 2‘ isomer, in accord with the published secondary phosphate dissociation constants (6, 14). The isomers of adenylic acid are not separable a t pH 6. The 3’ adenylic acid does not move faster than the 2‘ isomer a t pH 6, as expected from the net charges which are calculated from the reported dissociation constants ( 1 , d ) . An explanation for the lack of separation was found accidentally, when it was observed that these isomers are resolved a t a p H above 8, as in Figure 1,G. The secondary phosphate groups of both isomers are over 95% dissociated under these conditions and the faster movement by the 2’ isomer must be evidence of a steric difference between the isomers which opposes the slight difference in net charge. The net charge difference a t a p H below 8, which is expected from the pK values ( I , 4),does not cause the 3’ isomer to migrate faster than the 2’ isomer, owing t o the opposing steric effect evident in the difference in mobility above pH 8. The 2’- and 3’-guanylic acids do not separate a t pH 6 , but resolution does occur in 240 minutes a t 38 volts per cm. in the region of pH 3.8 in formate buffer of 0.1 ionic strength (Figure 1 , H ) . This resolution has also been reported by Davidson and Smellie ( 9 ) . SUMMARY
The four 3‘ ribonucleotides are resolved by electrophoresis in paper in 90 minutes under a field of 30 volts per em. in both
formate buffer of pH 3.5 and ionic strength 0.1 and 0.lM sodium tetraborate solution. Each 5’ isomer may be separated from the corresponding 2’ and 3’ isomers in 90 minutes with a field of 37 volts per em. in 0.1M sodium tetraborate solution. The 2’- and 3‘-cytidylic acids as well as the 2‘- and 3‘-uridylic acids may be resolved in 150 minutes under a field of 37 volts per em. in phosphate buffer of pH 5.8 and ionic strength 0.1. The 2’and 3’-adenylic acids are resolved in 120 minutes a t 40 volts per em. in 0.1 ionic strength buffers of pH greater than 8. The 2‘and 3’-guanylic acids are separated in 240 minutes a t 38 volts per em. in formate buffer of pH 3.8 and ionic strength 0.1. LITERATURE CITED
(1) ..klbertv. R . -4.. Smith. R. M.. and Bock. R. M.. J . Biol. Chem.. 193,-425 (1951). (2) Boeseken, J.,Advances in Curbohydrate Chem., 4, 189 (1949). (3) Boulanger, P., and Montreuil, P., Bull. soc. chim., France, 1952, \
,
844. (4) Carter, C. E., and Cohn, W. E., J . Am. Chem. Soc., 72, 2604
(1950). (5) Cavalieri,L. F., Ibid., 75, 5268 (1953). (6) Cohn, W. E., J . Cellular Comp. PhysioZ.,’38, Suppl. 1,21 (1961). (7) Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J., 40,33 (1946). (8) Crestfield, A. M., and Allen, F. W., ANAL.CHEM.,27, 422 (1955). (9) Davidson, J. N., and Smellie, R. M. S.,Biochem. J., 52, 599 (1952). (10) Hanes, C. S., and Isherwood, F. A., Nature, 164, 1107 (1949). (11) Jaenicke, L., and Vollbrechtshausen, I., Naturwissenschaften, 39,86 (1952). (12) Levene, P. A , , and Simms, H. S.,J . Biol. Chem., 65, 519 (1925); 70, 327 (1926). (13) Levene, P. A,. Simms, H. S., and Bass, L. W., Ibid., 70, 229, 243 (1926). (14) Loring, H. S., Bortner, H. W,, Levy, L. W., and Hammell, M. L., Ibid., 196,807 (1952). (15) Markham, R., and Smith, J. D., Biochem. J., 49, 401 (1951). (16) Markham, R., and Smith, J.D., Nature, 168,406 (1951). (17) Vischer, E., and Chargaff, E., J. Bid. Chem., 176, 703 (1948). (18) Werkheiser, W., and Winder, R., Ibid., 204, 971 (1953). (19) Wieland, T., and Bauer, L., Angew. Chem., 63, 512 (1951). (20) Zittle, C. 4.,Advances in Enzymol., 12, 493 (1951). RECEIVED for review September 24, 1954. Accepted November 24, 1954. Work supported in part b y Grant-in-Aid RG 2496, U. S. Public Health Service, and by Cancer Research Funds of the University of California.
A High Rate of Shear Rotational Viscometer E. M. BARBER, 1. R. MUENGER, and F. J. VILLFORTH, JR. Beacon Laboratories, The Texas Co., Beacon,
N. Y.
The assumption that \-iscosity is independent of shear rate is implicit in conventional viscometry, hut is not valid €or plastic materials nor for many fluids which are becoming increasingly important in lubrication and other fields. These materials should he studied over a wide range of shear rates. This paper presents information on the design, construction, and typical operation of a rotational viscometer for operation at shear rates up to about 1,000,000 reciprocal seconds. A simple and novel method was devised to deal with the heat generated at high rates of shear in the fluid film. The paper presents calibration data on the viscometer; some indications of the adequacy of the methods for handling the heating within the film; and sample data for several fluids, including two fluids that had been tested by another investigator; and suggests one possible simplification in describing or measuring the viscosity behavior of polymer-thickened oils.
V
ISCOSITY is one of the important properties that must be dealt 7%-ithin many liquid flow problems, particularly in bearing lubrication, where viscous forces determine both the load-carrying capacity and the friction of the bearing. Precise methods have been developed for measuring the viscosity of liquids. These methods generally have been developed around Kerton’e law of viscosity and his assumption that viscosity is independent of the rate of shear. The most widely used method comprises flow through a capillary (1, 2 ) ; rotational viscometers (3, 10, 12) are used much less frequently. Ordinarily, viscosity measurements are made a t relatively low rates of shear, which vary across the capillary, with respect to time during a determination, and from sample to sample depending upon the viscosity and density of the liquid being tested. Most pure liquids and mineral lubricating oils are Newtonian in the sense that their viscosity is independent of rate of shear. Many industrially important materials such as blends of mineral oils with polymers, paints, inks, and greases are non-h-ewtonian
