Due to this fact the concentration of free (hydrated) Mn2+ ions changes in the course of the reaction and, consequently, the intensity of their ESR signal also changes. ... The technique is based on the differential ability of NTP and the products.
ANALYTICAL
BIOCHEMISTRY
Continuous Biochemical
77, 413-418 (1977)
Electron Spin Resonance Detection of Reactions of Nucleoside Triphosphates J. M. BACKERANDI.
A. SLEPNJOVA
Institute of Chemical Kinetics and Combastion, Siberian Branch of the Soviet Academy of Sciences, Novosibirsk 630090, USSR Received January 14, 1976; accepted August 19, 1976 A technique of continuous recording of the kinetics of biochemical reactions of nucleoside triphosphates by means of an ESR (electron spin resonance) method is proposed. The technique is based on the differential ability of NTP (nucleoside triphosphates) and the products of their conversion to coordinate MnZ+ ions. Due to this fact the concentration of free (hydrated) Mn2+ ions changes in the course of the reaction and, consequently, the intensity of their ESR signal also changes. The proposed technique makes it possible to determine changes of concentration 20.1% of the total free ion concentration. The technique was applied to observation of reactions catalyzed by RNA polymerase, alkaline phosphatase, and aminoacyl-tRNA synthetase.
Many of the biochemical reactions of nucleoside triphosphates (NTP) are known. Necessary cofactors of such reactions are Mg2+ and/or Mn2+ ions. In this paper we propose a simple technique of continuous recording of such reactions by means of electron spin resonance (ESR) method. The technique is based on the differential ability of NTP and the products of their conversion to coordinate Mn2+ ions. Consequently, the concentration of free Mn2+ ions changes in the course of the biochemical reaction. This process can be detected by peak intensity changes in one of the superfine components of the esr signal of free (hydrated) Mn2+ ions. A schedule of continuous recording is proposed which makes it possible to determine changes of concentration ~0.1% of the total free ion concentration. In this paper we also describe examples of the ESR method as applied to observations of reactions catalyzed by RNA polymerase, alkaline phosphatase, and aminoacyl-tRNA synthetase. MATERIALS
AND METHODS
The following reagents were used: ATP (Reanal, Hungary), [3H]ATP, [14C]ATP (Amersham, England), GTP, CTP, UTP, poly(U) (SKTB BAC, Novosibirsk). ATP and [3H]ATP were repurified by column chromatography. DNA from T7 phage was kindly provided by M. A. Grachev (Institute of Organic Chemistry, Novosibirsk) and alkaline phosphatase (PME) by E. Zichikov (Institute of Organic Chemistry, Novosibirsk). 413 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN 0003-2697
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BACKER
AND
SLEPNJOVA
RNA polymerase, kindly provided by N. M. Pustoshilova (SKTB BAC, Novosibirsk), had the following characteristics: C = 4.64 mg/ml; 1 activity unit = 1.3 wg; A28,jA260 = 1.59; nonmatrix synthesis, 1.2%. Phenylalanine-tRNA synthetase (PRSase) from Escherichia coli was kindly provided by 0. I. Lavric (Institute of Organic Chemistry, Novosibirsk). Tryptophanyl-tRNA synthetase from bovine pancreas was kindly provided by 0. Favorova (Institute of Molecular Biology, Moscow). The recording of changes of free Mn2+ ion concentration in the course of the biochemical reaction was carried out in an ESR spectrometer (E-3 Varian, Palo Alto, Calif.). The preparation was drawn into a thermostated (CC&, 37°C). U-shaped capillary installed in the resonator of the ESR spectrometer. The volume of the sample ranged from 0.05 to 0.1 cm3. In the case of the RNA polymerase reaction, along with ESR recording, the synthesis rate was determined by a radiochemical method based on the incorporation of [14C]AMP into the acid-insoluble product. The radioactivity of 3-mm paper disks was measured in a liquid scintillation counter (Mark, Brockton, Mass.). Separation of products in the reactions catalyzed by PME and PRSase was carried out by paper chromatography on FN-3 (DDR) in a system of isobutyric acid-2.3 M NH40H (66:34). The ratio of the reaction products (ATP, ADP, and AMP) was determined on the basis of 3H labeling. RESULTS Measurement
Technique
When the biochemical reactions of NTP are carried out in standard reaction mixtures, the total concentration of ions of bivalent metals exceeds the total concentration of NTP. In such mixtures NTP coordinates a portion of ion complexes with stability constants of 10e4- 1O-5 M. The coordination of Mn2+ ions into asymmetrical complexes results in broadening the Mn2+ ESR spectrum components that cause significant (15-30 times) decreases in the peak intensity of superfine components of the spectrum. Consequently, when an excess of free Mn2+ ions is present, it is their ESR spectrum that is recorded. If the concentration of coordinated ions changes in the course of a biochemical reaction, it will lead to changes in the ESR spectrum intensity. To detect these changes we used the following technique. The intensity of the outer magnetic field (H,) was chosen to correspond to the position of the minimum (or maximum) of the second component of the superfine structure (SFS) in the ESR spectrum of free Mn2+ ions. An amplification factor of a 100-kHz amplifier of the ESR spectrometer was chosen so that the total amplitude of the second SFS component was bout lo-20 times larger than the maximal ordinate axis recorder pen deviation. With time scanning in operation, such deviation of the recorder pen
ESR DETECTION
OF NTP REACTIONS
41.5
2
FIG. 1. The change in concentration of bound MnZ+ ions in the synthesis of poly(A) on a poly(U) matrix. The reaction mixture contained: 0.04 M Tris-HCI, pH 7.8; 3 X 10e4 M poly(U); 1.5 x 10-3~ [WIATP; 5 x 10W~ of MnCI,; 0.28 mg/ml of RNA polymerase. Curve a: no GTP; curve b: 1.5 x 10m3M GTP (monomanganese salt). (0, A): Incorporation of [Y]AMP into a polymer product.
can be determined by the intensity change of one-half of the superfine component amplitude. To calibrate these intensity changes we use water or water-glycerin solutions of MnCl,. It is obvious that the sensitivity of the proposed technique is determined by the absolute sensitivity of the ESR spectrometer and by the limits of linearity of the lOO-kHz amplifier. In our experiments with the concentration of free ions (Mr$+) equal to l-3 x 10e3 M, we detected intensity changes corresponding to an Mn*+ concentration of 3 x lo+ M, the signal/ noise ratio being 5. Reactions Catalyzed By RNA Polymerase
In the course of such a reaction the pyrophosphate residue is separated from NTP, and the nucleoside monophosphate (NMP) thus formed is incorporated into a polymer chain. At the Mn2+ ion concentrations used, both NTP and the pyrophosphate residues formed exist in solution in the form of monomanganese complexes (1). The polymer products formed also coordinate MnZ+ ions with a stoichiometry of one ion to two monomeric residues in a polymer chain (2). This means that each act of incorporation into a polymer chain will be accompanied by the appearance of onehalf of the coordination site and, consequently, the intensity of the free ion ESR signal will fall. Figure 1 presents the kinetic curves for the RNA polymerase reaction on a poly(U) matrix in the presence of Mn2+ ions. As is shown, the increase in concentration of bound Mn2+ ions corresponds to the incorporation of radioactive label. and the conversion factor for the data obtained
416
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5
f0
6
min
FIG. 2. The change in concentration of bound Mn*+ ions in the synthesis of RNA on DNA from T7 phage. The reaction mixture contained: 0.04 M Tris-HCI, pH 7.8; 3 x 10e4 M DNA; 4 x 1OWM NTP; 4 x IOPM MnCl,; 1.2 x 10w2~ MgCl*; 0.09 r&ml of RNA polymerase. (0): Incorporation of [W]AMP into a polymer product.
by different methods is equal to 2, as is expected. This factor does not depend on the presence of uncomplementary NTP in the solution (Fig. 1, curve b). In the case of the RNA polymerase reaction of a DNA matrix it is difficult to predict the conversion factor because both Mn2+ and Mg2+ ions are present in the solution. However, as shown in Fig. 2, the
FIG. 3. The change in concentration of bound Mn *+ ions in the hydrolysis of ATP by alakline phosphatase. The reaction mixture contained: 3 x lo+ M ATP; 0.02 M Tris-HCl, pH 6.8; 5 x l(r3 M MnCl,; 0.3 mg/ml of protein.
ESR DETECTION
2
4
417
OF NTP REACTIONS
6
8
m
&
i4
miff
L
FIG. 4. The change in concentration of free Mnz+ tons in the reactions catalyzed by PRSase. One milliliter of the reaction mixture contained: (a), 3 pmol of ATP, 1 mg of tRNA, 2 nmol of phenylalanine, 3 mol of MnCl,; 50 /.UIIOIof Tris-HCI, pH 7.8; 0.2 mgofprotein; (b), 3 pm01 of ATP; 5 pmol of MnCl,; 50 pmol of Tris-HCl pH 7.8; 0.2 mg of protein; (c), in the plateau region of the kinetic curve, tRNA and phenylalanine were added to reaction mixture b; (d), same as in b plus 3 x 1O-3 M orthophosphate.
kinetic curve obtained by the ESR method is in agreement obtained by the radiochemical method.
with the data
The Reaction Catalyzed By Alkaline Phosphatase
Figure 3 presents the kinetic curve obtained by the ESR method for the hydrolysis of ATP catalyzed by PME. All products of the reaction form monomanganese complexes with stability constants less than that of ATP. However, because of the growth of total ligand concentration, the free MnZ+ ion concentration decreases. The comparison of the kinetic data with the results of chromatographic analysis of the reaction products indicates that additional coordination of one Mn2+ ion corresponds to the hydrolysis of eight to nine molecules of ATP to ADP.
418
Reactions
BACKER
AND SLEPNJOVA
Catalyzed by Aminoacyl-tRNA
Synthetase
Figure 4 presents a kinetic curve with a plateau region. The curve was obtained by the ESR method in the course of an aminoacylation reaction catalyzed by PRSase. It was found in the course of this reaction, in contrast to former ones, that the concentration of bound ions decreases (curve a). Chromatographic analysis indicated that the mixture after reaction contained 86% ATP, 11% ADP, and 3% AMP. Incubation of ATP with PRSase results in a similar kinetic curve (b), but in the course of this reaction, only ADP is formed. It should be noted that the substitution of Mn2+ by Mg2+ ions causes ADP formation as well. The addition of the PRSase substrates tRNA and phenylalanine in the plateau region resulted in a resumption of hydrolysis of ATP to ADP (curve c). Similar results were obtained for tryptophanyl-tRNA synthetase from bovine pancreas. Judging by the reaction products in the course of this reaction as well as in the case of PME hydrolysis, the concentration of bound ions should grow. The reverse trend of the kinetic curves can be explained if we suppose that either ADP or orthophosphate does not take part in the coordination of Mn2+ ions but is bound directly to enzymes. It was found that, after addition of orthophosphate to the reaction mixture, the shape of the kinetic curve corresponds to that expected for the hydrolysis of ATP to ADP and orthophosphate (curve d). In conclusion it should be noted that the technique of continuous recording proposed here can be applied not only to NTP reactions but to any reaction in the course of which a concentration change of paramagnetic particles possessing a certain ESR spectrum takes place. We hope that the high sensitivity, the small amounts of reagents involved, and the technical simplicity of this method will make it attractive to investigators. ACKNOWLEDGMENTS The authors thank 0. A. Anisimov and Professor Yu. N. Molin for helpful discussions.
REFERENCES 1. Stability Constants of Metal-Ion Complexes, Sect. l-2 (1964), Chemical Society, London. 2. Cohn, M., Danchin, A., Grunberg-Manago, M. (1969)J. Mol. Bio/. 39, 199-214.
