Kinetics of hydrogen-deuterium exchange in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) determined by laser-Raman spectroscopy

Abstract

Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in adenosine 5'-monophosphate, adenosine 3':5'-monophosphate, and poly(riboadenylic acid) (poly(rA)) were determined as a function of temperature in the range 20-90 degrees C by means of laser-Raman spectroscopy. For 5'-rAMP, the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature, i.e., kpsi = Ae-Ea/RT, with A = 2.3 X 10(14) hr-1 and Ea = 24.2 +/- 0.6 kcal/mol. For cAMP, above 50 degrees C, kpsi is nearly identical in magnitude and temperature dependence to that of 5'-rAMP. However, below 50 degrees C, isotope exchange in cAMP is much more rapid than in 5'-rAMP, characterized by a lower activation energy (17.7 kcal/mol) and frequency factor (9.6 X 10(9) hr-1). Exchange in poly(rA) is considerably slower than in 5'-rAMP at all temperatures, but like cAMP the in k vs. 1/T plot may be divided into high temperature and low temperature domains, each characterized by different Arrhenius parameters. Above 60 degrees C, poly(rA) gives Ea = 22.0 kcal/mol and A = 3.2 X 10(12) hr-1, while below 60 degrees C, Ea = 27.7 kcal/mol and A = 1.8 X 10(16) hr-1. Thus, increasing the temperature above 60 degrees C does not diminish the retardation of exchange in poly(rA) vis a vis 5'-rAMP. These results indicate that the distribution of electrons in the adenine ring of cAMP is altered by lowering the temperature below 50 degrees C, although no similar perturbation occurs for 5'-rAMP. Retardation of exchange in poly(rA) is most probably due to base stacking at lower temperatures and to steric hindrance from the ribopolymer backbone at higher temperatures. We also report the spectral effects of deuterium exchange on the vibrational Raman frequencies of 5'-rAMP, cAMP, and poly(rA) and suggest a number of new assignments for the 5' and cyclic ribosyl phosphate groups.

Interaction of Z form poly(dG-dC). poly(dG-dC) with divalent metal ions: localization of the binding sites by I.R. spectroscopy

“…Biochemicals. Specific deuteration of the guanines was obtained by incubation in D20 at 80°C for 3 hours (24)(25). Samples (Fig.…”

Dependence of purine 8C‐H exchange on nucleic acid conformation and base‐pairing geometry: A dynamic probe of DNA and RNA secondary structures

“…18 Briefly, Raman cells containing the polynucleo-tide were incubated at the temperature selected for exchange (50 k 0,l'C). Sample cells were removed periodically from the incubator and thermostated at low temperature in the Raman illuminator to effectively arrest exchange while spectral data were collected.…”

“…The observed temperature dependence of retardation in poly(rA) correlates well with the degree of base stacking computed from hydrodynamic measurements. 18 Detailed characteri~ationl~ of the equilibrium between poly(r1) and [poly(rI)], provides another fairly well defined system for semiquantitative correlation of the retardation of exchange with the mole percentage of purine residues incorporated into a single-stranded and stacked helical structure. Since the relative stabilities of the singlestranded and quadruplex structures are dependent on the nature of the alkali metal counterion, it is possible to define a family of aqueous poly(r1) salts that differ in the position of the equilibrium: 4poly(rl) s [p~ly(rI)]~.l~ The observed retardation factors for the polyinosinates, poly(r1) -NH,+ (100% single-stranded), poly(r1) -Na+ (90% ss), poly(r1) -Li+ (80% ss), and poly(r1).…”

The Synthesis of Deuterionucleosides

“…The pH-dependent kinetic studies of exchange process have also been performed 52,71,83,[85][86][87][88] . It has been found, that within a certain pH range, the structure of the purine could be unchanged (neither protonated nor deprotonated).…”

“…In case of N9 substituted purines the rate of the exchange reaction sharply increases at pH higher than 12 88 . Therefore, the reaction is usually carried out in a slightly alkaline (pH = 7.8) 52 or neutral media using the published procedures 87,88 . This method has been used for deuteration of C8 of guanosine triphosphate and guanosine monophosphate (incubation of the guanosine derivatives in 2 H 2 O at 50 ?C for 24 hours), and the level of deuterium incorporation is satisfactory for NMR studies of the RNA oligomer 49,53 .…”