Kinetics and equilibria in the acid denaturation of deoxyribonucleic acids from various sources

Abstract

SynopsisThe acid deiisturat,ioii trf :I vsriet,y of 1)NAl samples wit.11 guanine -cytosine (GC) conte~its ranging from 3744% hns been investigated by means of potentiometric, spectrophot,ometric, and thermal titrations, including spect.rophotonietric titrations in a stopped-flow fast reaction apparatus. As the pH of a solution of native UN.4 is lowered from the neutral range the net charge oil the mylecule becomes more positive as a result of the addition of protons to the bases adeuine, cytosine, and to a lesser extent guanine. At a fairly sharply defined value of the charge a conformational transition sets in which is completed after the charge has increased in the positive direction by aii additional 0.4-0.5 unit per base pair. The threshold value of the charge is dependent on the composition of the DNA as well as various experiineiital parameters such a s temperature and ioiiic strength. At 25°C. ill 0.lM NaCl the threshold value is approximat,ely 0.55 charge unit per base pair relative t,o pH 6 for a GC coiiterit of 3T%,, and 0 . i X for 647;, CG. The conformational transition is accompanied by heat. absorption amounting, in the case of salmon DNA in 0.1M NaCl, to 3020 f 150, 4920 f 200, and 5700 f 230 cal./mole of base pairs at 5, 25, and 45"C., respectively. If it is assumed that the heats of ionization of t,he bases are the same in denatured DNA a 5 in the isolated nucleotides, t.he result at 25" C. can be corrected by using previously determined heats of ionizatiou t o give for the hypothetical process DNA (native, p H 6) + DNA (denatured, p H 6) AH = 8300 cal./mole of base pairs. A H appear@ to be independent of DNA composition, an average value of 8000 ca1Jmole of base pairs being obtained for DNA samples having GC contents ranging from 37-64%. The calorimetric results are consistent with the view that hydrophobic interactions are important in the stabilization of the DNA double helix. The kinetics of the acid denaturation of salmon DNA is complex.* This rmearch was aided by grants from {,he National Institutes of Health of the U.S. Public Health Service (CA 05007, HD 050U7 and GM 04725) and the National Science Foundation (HU 9625). E. P. G. gratefully acknowledges a suinmer fellowship of the Lalor Foundation, held a t the time these experiment8 were begun. t Present address: Biology Division, Argonne National Laboratmy, Argorme, Illiuois.1 Abbreviat.ions employed in this paper: DNA, deoxyribonucleic acid; RNA, riboraicleic acid ; AT, adeniiie-thymine; GC, giiaiiiiieryt.osiiie; poly A, polyriboadenylic acid; poly U, polyriboiiridylic acid; poly (A + U), 1: 1 complex of poly A and poly U; dAMP, deoxyadenylic acid; dCMP, deoxycytidylic acid; dGMP, deoxyguanylic acid; AdR, adenine deoxyriboside; CdR, cytosine deoxyriboside. 21 3 214 BUNVILLE, E T AL.The reaction is composed of an initial phase too rapid to be followed in the stoppedflow spectrophotometer and a slower phase. The half-time of the slower phase decreases with decreasing pH and increasing temperature, the latter decrease corresponding to an a...

Theoretical and Experimental Study of DNA Helix-Coil Transition in Acidic and Alkaline Medium

“…This increase is small for individual nucleotides and denaturated DNA It is more significant for helical DNA characterized by high charge density. pK 2 G = 3.7 was determined for O.lM NaCl in (14). In the present study, the concentration of N a+ is equal to 0.195M.…”

“…In (12,13) the pK value of cytosine was estimated directly in melted DNA regions. One can estimate the pK 2 G (pK of guanine of bonded base pairs) using the results of(l2, 14). According to them pK 2 G-3.7 (index 1 relates to melted regions, and index 2 relates to helical ones).…”

The Physical and Chemical Properties of Nucleic Acids

“…This is done most directly by heat-capacity measurements, which have not yet been extensively employed (153). The most thorough studies are those of Bunville et al (108) the acid denaturation of DNA. It is necessary to take into account the contribution of the heat of ionization of the bases in determining the heat of DNA denaturation by acid.…”

Equilibrium Studies on the Formaldehyde Reaction with Native DNA

“…Recent calorimetric measurements made by Bunville et af. [17] and also by Privalov et ul. [18] showed that the average value of A H for denaturation was close to 9 kcal/mol base pairs (37.6 kJ/mol) at neutral pH.…”