The natural polyamine spermine functions directly as a free radical scavenger

Abstract

The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.

Figure 1

Protection of ROS-induced DNA damage by spermine. ΦX-174 plasmid DNA (200 ng) was incubated in the ROS-generating system. (A) Where indicated, spermine, 1 mM catalase (500 units/ml) or reduced glutathione (4 mM). (B) Protective effects of increasing concentrations of spermine. O, open circle; L, Linear; SC, supercoiled DNA.

Figure 2

Effects of spermine on the hydroxyl radical spin-trapping by DMPO in the ROS-generating system. (A) Upper EPR spectrum is from a sample containing 30 μM H₂O₂ + 10 μM Cu(II). Lower EPR spectrum is from a sample containing 30 μM H₂O₂ + 10 μM Cu(II) + 1 mM spermine. Incubation of H₂O₂ without Cu(II) or the addition of bathocuproinedisulfonic acid, a Cu(I) chelator, to the H₂O₂/Cu(II) mixture completely inhibited the formation of the DMPO—OH quartet (not shown). These results are representative of at least three sets of experiments. (B) Effects of increasing concentrations of spermine on DMPO—OH formation in the ROS-generating system.

Figure 3

(a). Synthetic scheme for bis-α-[¹³C]spermine. 1,10-Bis([¹³C]Cyano)-3,8-diazadecane (4). A 0.198-g portion of 1,4-diaminobutane (2) (0.00224 mol) was added to a solution of 0.250 g (0.00465 mol, 2.07-fold excess) of [¹³C]cyano-enriched acrylonitrile (3) in 6 ml of ethanol, and the reaction was allowed to reflux for 18 hr. The solvent was then removed under reduced pressure, and the dark brown, oily residue was purified on silica gel (4 × 8 cm column, CHCl₃:MeOH:NH₄OH 900:600:3) to afford 0.261 g of a pale yellow oil, with an R_f of 0.41 (59.3% yield). ¹H-NMR (CDCl₃, ppm δ 1.54 (t, 4h, H₅ and H₆), 2.10 (s, 1H, NH), 2.56 (q, 4H, H₄ and H₇), 2.65 (t, 4H, H₂ and H₉), 2.91 (t, 4H, H₁ and H₁₀); IR cm⁻¹ 3306 (NH), 2938 (aliphatic), 2198 (CN); ¹³C-NMR CDCl₃, ppm δ 118.6 (CN). Bis(δ-¹³C)-spermine (1). A 0.261-g portion of (4) and 0.267 g of NaOH were added to a suspension of 0.458 g of Raney nickel in 20 ml of dry ethanol, and the mixture was hydrogenated (Parr apparatus) at 50 psi for 24 hr. The reaction was then filtered (Zetapore 0.45 δ) and the solvent was removed under reduced pressure. The yellow solid residue was dissolved in 30 ml of water and washed with three 25-ml portions of chloroform. The aqueous layer was then acidified to pH 1, and the solution was concentrated under reduced pressure to afford 0.56 g of a white solid. Recrystallization of this solid from water/ethanol afforded 0.41 g (89.1%) of bis-(α-¹³C)-spermine (1) as the tetrachloride salt. ¹H-NMR (D₂O, ppm) δ = 1.77 (t, 4h, H₆ and H₇), 2.08 (m, 4H, H₂ and H₁₁), 3.11 (t, 12H, H₁, H₃, H₅, H₈, H₁₀ and H₁₂); ¹³C-NMR (D₂O, ppm) δ = 36.5 (¹³CH₂—NH₃). (b) NMR data for [¹³C]spermine free radical scavenging experiments. Spectrum A: bis-α-¹³C-enriched spermine. Spectrum B: 1% TSP in D₂O. Spectrum C: 1 mM bis-α-¹³C-enriched spermine plus 0.1 M H₂O₂, 33.3 mM CuCl₂. Spectrum D: 0.5 mM bis-α-¹³C-enriched spermine plus 0.5 M H₂O₂, 167 mM CuCl_2. All spectra were recorded on a General Electric QE 300 mHz FT-NMR, in D₂O.

Figure 4

Mass spectral analysis for [¹³C]spermine free radical scavenging experiments. (A) CI spectrum of bis-α-[¹³C]-enriched spermine standard. (B) CI mass spectral analysis of unknown products of free radical scavenging reaction. Mass spectroscopy was performed on a VG 7250S MicroMass spectrometer (MicroMass Corp., Manchester, U.K.) equipped with a direct chemical ionization (DCI) probe and using ammonia as the reagent gas.

Figure 5

Proposed mechanism for hydroxyl radical scavenging by spermine that is consistent with EPR, NMR, and MS results. Compound (1), bis-α-[¹³C]spermine; compound (5), bis-α-1-(N,N-dihydroxy)amino-12-(N,N-dihydroxy)amino-4,9-diaza[¹³C]dodecane; compound (6), 1,12-bis-1,12-di-(hydroxyimino)-4,9-diaza[¹³C]dodecane; compound (7), 1,12- bis-1,12-dioxo-4,9-diaza[¹³C]dodecane.