RAMAN-SPECTROSCOPY OF DNA-METAL COMPLEXES .2. THE THERMAL-DENATURATION OF DNA IN THE PRESENCE OF SR2+, BA2+, MG2+, CA2+, MN2+, CO2+, NI2+, AND CD2+

Differential scanning calorimetry, laser Raman spectroscopy, optical d ensitometry, and pH potentiometry have been used to investigate DNA me lting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg 2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M(2+)]/[PO2-] = 0.6 in aqueous solution s containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. A ll of the alkaline earth metals, plus Mn2+, elevate the melting temper ature of DNA (T-m > 75.5 degrees C), whereas the transition metals Co2 +, Ni2+, and Cd2+ lower T-m. Calorimetric (Delta H-cal) and van't Hoff (Delta H-VH) enthalpies of melting range from 6.2-8.7 kcal/mol bp and 75.6-188.6 kcal/mol cooperative unit, respectively, and entropies fro m 17.5 to 24.7 cal/K mol bp. The average number of base pairs in a coo perative melting unit (<n(melt)>) varied from 11.3 to 28.1. No dichoto my was observed between alkaline earth and transition DNA-metal comple xes for any of the thermodynamic parameters other than their effects o n T-m. These results complement Raman difference spectra, which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after therm al denaturation. Raman difference spectroscopy shows that transition m etals interact with the N7 atom of guanine in duplex DNA. A broader ra nge of interaction sites with single-stranded DNA includes ionic phosp hates, the N1 and N7 atoms of purines, and the N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, whereas spectra of single-stranded complexes are very simi lar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations at 1092 cm(-1) (nPO(2)(-)), 1 258 cm(-1) (dC, dA), and 1668 cm(-1) (nC=O, dNH(2) dT, dG, dC). Increa sed spectral intensity could also be observed near 1335 cm(-1) (dA, dG ) for CaDNA. Optical densitometry, employed to detect DNA aggregation, reveals increased turbidity during the melting transition for all div alent DNA-metal complexes, except SrDNA and BaDNA. Turbidity was not o bserved for DNA in the absence of metal. A correlation was made betwee n DNA melting, aggregation, and the ratio of Raman intensities I-1335/ I-1374. At room temperature, DNA-metal interactions result in a pH dro p of 1.2-2.2 units for alkaline earths and more than 2.5 units for tra nsition metals. Sr2+, Ba2+, and Mg2+ cause protonated sites on the DNA to become thermally labile. These results lead to a model that descri bes DNA aggregation and denaturation during heating in the presence of divalent metal cations: 1) The cations initially interact with the DN A at phosphate and/or base sites, resulting in proton displacement. 2) A combination of metal-base interactions and heating disrupts the bas e pairing within the DNA duplex. This allows divalent metals and proto ns to bind to additional sites on the DNA bases during the aggregation /melting process. 3) Strands whose bases have swung open upon disrupti on are linked to neighboring strands by metal ion bridges. 4) Near the midpoint of the melting transition, thermal energy breaks up the aggr egate. We have no evidence to indicate whether metal ion cross-bridges or direct base-base interactions rupture first. 5) Finally, all cross -links break, resulting in single-stranded DNA complexed with metal io ns.