Orientation of iron bleomycin and porphyrin complexes on DNA fibers

Bleomycin (Blm) is an antitumor agent that requires iron and oxygen for strand cleavage of DNA. In this study, ferric bleomycin, Fe(III)Blm, or the nitric oxide adduct of ferrous bleomycin, ON-Fe(II)Blm, were bound to one-dimensionally oriented DNA fibers. Reductive nitrosylation of Fe(III) complexes took place in situ on B-form DNA fibers. Electron paramagnetic resonance (EPR) spectra were obtained as a function of the angle phi between the magnetic field B and the fiber axis Zf. For comparison, EPR spectra were acquired for ON-Fe(II)TMpyP and ON-Fe(II)TMpyP-Im on oriented DNA fibers, where TMpyP is 5,10,15,20-tetrakis(1-methyl-4-pyridino)porphyrin and Im is imidazole. EPR spectra showed both low-spin Fe(III)Blm and ON-Fe(II)Blm bound to B-form DNA in two slightly different binding orientations in the ratio of 1:0.2. With A-form DNA, a fraction of bound Fe(III)Blm was high spin. Specifically, the angle beta between the fiber axis Zf and the g axis, gz, perpendicular to or nearly perpendicular to the equatorial plane of the iron complex was estimated as 20 degrees and 25 degrees for ON-Fe(II)Blm and 30 degrees and 25 degrees for Fe(III)Blm, respectively. The angle gamma that determines the orientation of gx and gy axes was estimated as 90 degrees for the two ON-Fe(II)Blm species and 10 degrees for the two Fe(III)Blm species, respectively. The NO was held rigidly in place as the temperature increased from 123 K to room temperature for ON-Fe(II)Blm but not for ON-Fe(II)TMpyP or ON-Fe(II)TMpyP-Im. It is hypothesized that the NO is structurally oriented by hydrogen bonding like the peroxide is held in HO2(-)-Co(III)Blm (Wu et al. J. Am. Chem. Soc. 1996, 118, 1281-1294). The EPR parameters are consistent with a six-coordinate complex for ON-Fe(II)Blm, although the superhyperfine structure from the trans nitrogen was not detected. The increase in g value anisotropy upon binding ON-Fe(II)Blm to DNA fiber may be caused by an increase in the overlap of d pi and 2p pi* orbitals induced by an interaction of NO with DNA and/or by a perturbation of d orbitals due to the pyrimidine-guanine interaction. It is concluded that the EPR parameters of ON-Fe(II)Blm and Fe(III)Blm bound to oriented DNA support the hypothesis that FeBlm species bind to DNA with adduct structures similar to those formed by related CoBlm species and DNA.     

TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline), a common fluorescent sensor for cellular zinc, images zinc proteins

Zn(2+) is a necessary cofactor for thousands of mammalian proteins. Research has suggested that transient fluxes of cellular Zn(2+) are also involved in processes such as apoptosis. Observations of Zn(2+) trafficking have been collected using Zn(2+) responsive fluorescent dyes. A commonly used Zn(2+) fluorophore is 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ). The chemical species responsible for TSQ's observed fluorescence in resting or activated cells have not been characterized. Parallel fluorescence microscopy and spectrofluorometry of LLC-PK(1) cells incubated with TSQ demonstrated punctate staining that concentrated around the nucleus and was characterized by an emission maximum near 470 nm. Addition of cell permeable Zn-pyrithione resulted in greatly increased, diffuse fluorescence that shifted the emission peak to 490 nm, indicative of the formation of Zn(TSQ)(2). TPEN (N,N,N'N'-tetrakis(-)[2-pyridylmethyl]-ethylenediamine), a cell permeant Zn(2+) chelator, largely quenched TSQ fluorescence returning the residual fluorescence to the 470 nm emission maximum. Gel filtration chromatography of cell supernatant from LLC-PK(1) cells treated with TSQ revealed that TSQ fluorescence (470 nm emission) eluted with the proteome fractions. Similarly, addition of TSQ to proteome prior to chromatography resulted in 470 nm fluorescence emission that was not observed in smaller molecular weight fractions. It is hypothesized that Zn-TSQ fluorescence, blue-shifted from the 490 nm emission maximum of Zn(TSQ)(2), results from ternary complex, TSQ-Zn-protein formation. As an example, Zn-carbonic anhydrase formed a ternary adduct with TSQ characterized by a fluorescence emission maximum of 470 nm and a dissociation constant of 1.55 × 10(-7) M. Quantification of TSQ-Zn-proteome fluorescence indicated that approximately 8% of cellular Zn(2+) was imaged by TSQ. These results were generalized to other cell types and model Zn-proteins.     

A new mixed integer program and extended look-ahead heuristic algorithm for the block relocation problem

This paper considers the block relocation problem (BRP), in which a set of identically-sized items is to be retrieved from a set of last-in-first-out (LIFO) stacks in a specific order using the fewest number of moves. The problem is encountered in the maritime container shipping industry and other industries where inventory is stored in stacks. After surveying the work done on the BRP, we introduce “BRP-III”—a new mathematical formulation for the BRP—and show that it has considerably fewer decision variables and better runtime performance than the other formulation in the literature. We then introduce a new look-ahead algorithm (LA-N) that is an extension of the algorithms from the literature and show that the new algorithm generally obtains better solutions than the other algorithms and has minimal CPU runtime.     

FREE RADICALS FROM THE PHOTODECOMPOSITION OF BLEOMYCIN

Abstract— Irradiation of bleomycin with light (λ > 320 nm) leads to a decrease in absorbance at 290 nm, which is suppressed by metal ions and by oxygen. Light-induced oxygen consumption is diminished by the enzymes superoxide dismutase and catalase, implying that toxic reduced species of oxygen (O₂ and H₂O₂) are formed during irradiation. Spin-trapping measurements with 5,5-dimethyl-1-pyrroline-1-oxide and 2-methyl-2-nitrosopropane demonstrated that hydroxyl radical and methyl radical adducts also are generated in the system. In addition, direct ESR measurements have shown that methyl radicals are produced during irradiation of bleomycin solutions at low temperatures, together with radicals probably derived from the bithiazole moiety of the bleomycin. The latter are also produced from irradiation of the model compound bithia. Radical production is diminished by complexation of bleomycin with metal ions.     

Resonance Raman studies of HOO-Co(III)bleomycin and Co(III)bleomycin: identification of two important vibrational modes, nu(Co-OOH) and nu(O-OH)

Bleomycin is an antitumor agent whose cytotoxicity is dependent on its ability to bind DNA in the nucleus and effect double-stranded DNA cleavage, which is difficult for the cell to repair. In order for this DNA cleavage to occur, bleomycin must, through a series of reactions, form a low-spin Fe(III) complex, the putative "activated" form of the drug, HOO-Fe(III)bleomycin. The relative strengths of the bonds in the Fe(III)-OOH linkage have not been determined due to the weakness of the hydroperoxo-to-iron(III) charge-transfer transition. The much more stable HOO-Co(III)bleomycin has often been studied as a structural analogue of HOO-Fe(III)bleomycin, and hence, an understanding of the relative bond strengths in the Co-OOH linkage may serve to enhance our understanding of the analogous Fe-OOH linkage. In this report, we present resonance Raman data that identify two important vibrational modes in the Co-OOH linkage, the stretching modes, nu(Co-OOH) and nu(O-OH). Both of these vibrational modes were found to be unperturbed by complexation of the drug with calf thymus DNA. Advantage was also taken of the isostructural realtionship between Fe-bleomycin and Co-bleomycin to analyze and assign the high-frequency modes for HOO-Co(III)bleomycin and Co(III)bleomycin (A(2) and B(2)). These data could be useful for future studies of photoactivated Co-bleomycin and Co-bleomycin analogues in an attempt to characterize oxygen-independent DNA damage pathways.     

Structure-reactivity relationships among metallothionein three-metal domains: role of non-cysteine amino acid residues in lobster metallothionein and human metallothionein-3

Metallothionein (MT) domains of different origins, exhibiting distinct, highly conserved cysteine positions, show differences in metal-cysteine coordination and reactivity. Lobster MT, which includes two Cd3S9 beta domains, was chosen as a basic model to study the structure-function relationship among the clusters. The possible influence of (1) the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters have been examined with the native lobster beta C and beta N domains, each having nine cysteines and binding three M2+ ions, and a modified domain beta C-->N, in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain. Each has been synthesized and characterized by UV, CD, 113Cd NMR, and 1H NMR spectroscopies. The synthetic native domains (Cd3 beta C and Cd3 beta N) displayed spectroscopic properties, metal-binding affinities, and kinetic reactivity similar to those of the holo protein. In contrast, the modified Cd3 beta C-->N domain was unusually reactive and, in the presence of Chelex, a metal-ion chelating resin, was converted to a Cd5(beta C-->N)2 dimer. These differences in structure and reactivity demonstrate that the requirements for formation of a stable type-B, Cd3S9, beta cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids. Molecular mechanics calculations suggest that changes of even a single amino acid in lobster Cd3 beta N toward lobster Cd3 beta C-->N or in mammalian MT1 or MT2 toward Cd3 beta-MT3 (GIF) can destabilize their structures.     

Reactions of the fluorescent sensor, Zinquin, with the zinc-proteome: adduct formation and ligand substitution

Zinquin (ZQ) is a commonly used sensor for cellular Zn(2+) status. It has been assumed that it measures accessible Zn(2+) concentrations in the nanomolar range. Instead, this report shows a consistent pattern across seven mammalian cell and tissue types that ZQ reacts with micromolar concentrations of Zn(2+) bound as Zn-proteins. The predominant class of products were ZQ-Zn-protein adducts that were characterized in vivo and in vitro by a fluorescence emission spectrum centered at about 470 nm, by their migration over Sephadex G-75 as protein not low molecular weight species, by the exclusion of reaction with lipid vesicles, and by their large aggregate concentration. In addition, variable, minor formation of Zn(ZQ)(2) with a fluorescence band at about 490 nm was observed in vivo in each case. Because incubation of isolated Zn-proteome with ZQ also generated similar amounts of Zn(ZQ)(2), it was concluded that this species had formed through direct ligand substitution in which ZQ had successfully competed for protein-bound Zn(2+). Parallel studies with the model Zn-proteins, alcohol dehydrogenase (ADH), and alkaline phosphatase (AP) revealed a similar picture of reactivity: ZQ(ACID) (Zinquin acid, (2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate)) able to bind to one Zn(2+) and extract the other in Zn(2)-ADH, whereas it removed one Zn(2+) from Zn(2)-AP and did not bind to the other. Zinquin ethyl ester (ethyl(2-methyl-8-p-toluenesulfonamido-6-quinolyloxy)acetate); ZQ(EE)) bound to both proteins without sequestering Zn(2+) from either one. In contrast to a closely related sensor, 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ), neither ZQ(ACID) nor ZQ(EE) associated with Zn-carbonic anhydrase. A survey of reactivity of these sensors with partially fractionated Zn-proteome confirmed that ZQ and TSQ bind to distinct, overlapping subsets of the Zn-proteome.     

Reaction of metal-binding ligands with the zinc proteome: zinc sensors and N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine

The commonly used Zn(2+) sensors 6-methoxy-8-p-toluenesulfonamidoquinoline (TSQ) and Zinquin have been shown to image zinc proteins as a result of the formation of sensor-zinc-protein ternary adducts not Zn(TSQ)(2) or Zn(Zinquin)(2) complexes. The powerful, cell-permeant chelating agent N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) is also used in conjunction with these and other Zn(2+) sensors to validate that the observed fluorescence enhancement seen with the sensors depends on intracellular interaction with Zn(2+). We demonstrated that the kinetics of the reaction of TPEN with cells pretreated with TSQ or Zinquin was not consistent with its reaction with Zn(TSQ)(2) or Zn(Zinquin)(2). Instead, TPEN and other chelating agents extract between 25 and 35% of the Zn(2+) bound to the proteome, including zinc(2+) from zinc metallothionein, and thereby quench some, but not all, of the sensor-zinc-protein fluorescence. Another mechanism in which TPEN exchanges with TSQ or Zinquin to form TPEN-zinc-protein adducts found support in the reactions of TPEN with Zinquin-zinc-alcohol dehydrogenase. TPEN also removed one of the two Zn(2+) ions per monomer from zinc-alcohol dehydrogenase and zinc-alkaline phosphatase, consistent with its ligand substitution reactivity with the zinc proteome.