Synthesis and structure-function study of artificial nucleases on the basis of hemin conjugates with peptide fragments of cell differentiation factor HLDF

A series of octa-hexapeptide fragments of HLDF and their conjugates with hemin were obtained by solid phase peptide synthesis. A relationship between the structure and the nuclease activity of the compounds was established. The effect of various factors (medium pH, the presence of metal ions, complexons, reducers, and buffer composition) on the DNA destruction with hemin peptides was studied. Preliminary information confirming an oxidative mechanism of this process was obtained. The cleavage of plasmid DNA under the action of hemin peptides was studied by the methods of electron microscopy, gel electrophoresis, and atomic force microscopy.     

Influence of hemin on the conformation of cyanogen bromide-cleaved peptides of apomyoglobin

The complexes of the three BrCN-cleaved fragments of sperm whale apomyoglobin with hemin were studied by circular dichroism (CD). In native myoglobin, the heme is located in the middle fragment; the isolated peptide (residues 56–131), however, produces little extrinsic Cotton effects by the addition of hemin, although about four molecules of hemin are bound to this peptide. In marked contrast, the COOH-terminal peptide (residues 132–153), which binds three hemin molecules, shows strong Cotton effects in the Soret bands and drastically changes its conformation from unordered to highly helical. The Arg-modified or Lys-deaminated peptide no longer undergoes conformational changes by the addition of hemin, suggesting that the two propionic acid groups of one hemin molecule interact with the Arg residue and one of the Lys residues, which stabilizes the induced helical conformation. The NH₂-terminal peptide (residues 1–55) binds one hemin molecules, and the helicity of this fragment is slightly enhanced by the addition of hemin. Both the CD and difference absorption spectra indicate that the mode of interaction between the peptides and hemin are different for the three apomyoglobin fragments.     

Binding reaction of hemin to globin

Binding of hemin to globin was studied in the presence of 25 mM caffeine by measuring CD and optical absorption changes in the Soret region. CD and optical absorption spectra after mixing equimolar amounts of hemin and globin were the same as those of ferric hemoglobin. In contrast, addition of excess globin to hemin formed a complex that was distinguishable from ferric hemoglobin in terms of the CD and optical absorption spectra. By comparing the spectra of the complex with those of various hemoglobin derivatives, it was concluded that the complex was globin which carried a hemin exclusively on the alpha chain. This means that the alpha chain of the globin molecule has a greater affinity for hemin than the beta chain, as observed by other investigators using hemin-cyanide. The rate of binding of hemin to globin was estimated by the use of CD and optical absorption stopped-flow apparatus. The rate of hemin binding to the alpha chain of globin was obtained by mixing hemin and excess globin, and that to the beta chain was obtained by mixing equimolar concentrations of hemin and globin. The results showed that hemin was bound to the alpha chain in the globin molecule to form a transient intermediate, followed by its transformation into another intermediate, the transformation was the rate-limiting step, and the beta chain in the globin molecule had a greater affinity for hemin after hemin binding to the alpha chain than before.     

Detection of beta-amyloid peptide aggregation using DNA electrophoresis

DNA could readily associate with the aggregated forms of the beta-amyloid peptides beta(1-40) and beta(25-35), giving rise to a shift in the electrophoretic mobility of DNA. As a result, DNA was retained at the top of a 1% agarose gel. In contrast, the electrophoretic mobility of DNA was little influenced by the monomeric forms of beta(1-40) and beta(25-30). DNA from different sources such as lambda phage, Escherichia coli plasmid, and human gene showed similar results. However, the electrophoretic mobility of RNA was shifted by the monomeric beta(1-40) and beta(25-35) as well as by the aggregated beta(1-40) and beta(25-35). The association of DNA with the aggregated beta-amyloid peptides could occur at pH 4-9. The inhibitory action of hemin on beta-amyloid aggregation could be confirmed using the DNA mobility shift assay. These results indicate that the DNA mobility shift assay is useful for kinetic study of beta-amyloid aggregation as well as for testing of agents that might modulate beta-amyloid aggregation.     

The interaction of hemin with skeletal muscle actin

The ability of actin to interact with hemin was studied. It was found that the Soret absorption band of hemin changes in the presence of actin and that hemin is capable of quenching the fluorescence intensity of actin. These findings were indicative of hemin binding to actin. The binding constant for the high affinity site was calculated to be 5.3 X 10(6) M-1. The amounts of native G- and F-actin were estimated by their DNAase I inhibition activity. It was observed that the binding of hemin to G-actin is followed by a slow decrease in the ability of actin to inhibit DNAase I activity and to polymerize upon addition of salts. Binding of hemin to F-actin resulted in a gradual depolymerization of the filaments, to an inactivated form, as expressed by a reduction in the ability of hemin-bound F-actin to inhibit DNAase I activity in the absence as well as in the presence of guanidine-HCl. Electron microscopy studies further corroborated these findings by demonstrating that: (1) hemin-bound G-actin failed to show formation of polymers when salts were added; (2) a marked reduction in the amount of actin polymers was observed in the specimens examined 24 h after mixing with hemin. It is suggested that the elevated amounts of free hemin formed under pathological conditions, might be toxic to cells by interfering with actin polymerization cycles.     

Determination of genome size, macrorestriction pattern polymorphism, and nonpigmentation-specific deletion in Yersinia pestis by pulsed-field gel electrophoresis.

Of 16 restriction endonucleases known to hydrolyze rare 6- or 8-base recognition sequences that were tested, only SpeI, NotI, AscI, and SfiI generated fragments of chromosomal DNA from Yersinia pestis, the causative agent of bubonic plague, of sufficient length to permit physical analysis by pulsed-field gel electrophoresis (PFGE). Of the individual bands detected after single-dimensional PFGE of these digests, the largest sum was obtained with SpeI (3,575.6 +/- 114.6 kb). Of these 41 bands, 3 were found to contain comigrating fragments, as judged by the results of two-dimensional SpeI-ApaI PFGE; addition of these fragments and the three plasmids of the species yielded a refined estimate of 4,397.9 +/- 134.6 kb for the genome. This size was similar for eight strains of diverse geographical origin that exhibited distinct DNA macrorestriction patterns closely related to their biotypes. The high-frequency chromosomal deletion known to exist in nonpigmented mutants (unable to assimilate Fe3+ at 37 degrees C or store hemin at 26 degrees C) was shown by two-dimensional PFGE analysis with SpeI and ApaI or with SfiI and SpeI to be 92.5 and 106 kb in size, respectively. The endpoints of this deletion were precise, and its size was more than sufficient to encode the eight known peptides reported to be absent in nonpigmented mutants. This deletion had not occurred (but was able to do so) in a rare mutant capable of hemin storage but not iron transport.     

Interaction of hemin with placental glutathione transferase

To verify a possible involvement of glutathione transferase pi in intracellular transport of hemin the interaction between the protein and the ligand was studied using three different spectroscopic techniques: intrinsic fluorescence quenching, kinetic measurements in the visible range and circular dichroism. From fluorescence experiments two binding sites for the hemin were found with Kd values of about 20 nM (high-affinity site) and 400 nM (low-affinity site). In the presence of glutathione or S-methylglutathione the high-affinity site further increased its affinity, while the second site reduced its affinity for hemin. The effect of hemin on the catalytic activity of the glutathione transferase pi was studied using two different glutathione concentrations. With 1 mM glutathione a non-linear Dixon plot was obtained, while decreased hemin inhibition and a linear pattern was observed with 2.5 mM glutathione. The Ki calculated was 4 microM and the inhibition appeared to be non-competitive with respect to 1-chloro-2,4-dinitrobenzene. CD spectra of the bilirubin-glutathione-transferase complex (350-600 nm region) at different hemin concentrations showed a common binding site for bilirubin and hemin. In conclusion, the presence of a high-affinity site for the hemin and the fact that glutathione at physiological concentrations increased the affinity of this site, suggest the involvement of glutathione transferase pi in the hemin transport.     

Effect of treatment with hemin on rat liver catalase.

Rats were injected with a single or repeated doses of hemin intraperitoneally, and the effect on liver catalase [EC 1.11.1.6] was studied. A single administration of hemin caused a reduction in the concentration of liver catalase, both in enzymatic activity and in catalase protein determined immunochemically. The reduction occurred a few hours after the hemin injection, and is probably due to stimulated degradation. Disappearance of radioactivity from liver catalase prelabelled with [14C]leucine was enhanced following the administration of hemin. No evidence for a repression in vivo incorporation of [14C]leucine and [3H]sigma-aminolevulinic acid into liver catalase was obtained with hemin-treated rats. When the hemin was given repeatedly at 12-h intervals, the level of liver catalase decreased considerably. However, the impairment in catalase-synthesizing activity of liver cells of rats thus treated was rather slight, when examined in a cell-free system. Some differences were noted between the results in the present study and those in previous investigations with Sedormid-treated rats.     

Inhibition of cancer cell invasiveness by synthetic peptides GEGEEGEE and DFGEEAEE

The ability of two synthetic peptides GEGEEGEE and DFGEEAEE to inhibit the invasiveness of tumor cells has been studied. Using confocal microscopy, it was demonstrated that these peptides specifically bind to receptor for hyaluronic acid mediated motility (RHAMM) on the prostate cancer cell surface. Effect of peptides on invadopodia formation in cancer cells was studied by fluorescent gelatin degradation. Using confocal microscopy, images of the cells were obtained and the degree of their invasiveness was analyzed by assessing the gelatin degradation area using ImageJ software. It was found that pre-incubation of tumor cells with the peptides at a concentration of 40 μg/mL (2 × 10^–7 M) inhibited the invasiveness by more than 80%. Thus, it was shown that peptides GEGEEGEE and DFGEEAEE may be potential antitumor agents.     

Interaction of hemin with quadruplex DNA

A DNA enzyme with peroxidase activity is a G-quadruplex-based DNAzyme formed by hemin and G-quadruplex DNA. Activity of peroxide DNAzymes can be influenced by the structure of quadruplex DNA. In this investigation, the interaction of hemin with T30695 G-quadruplex DNA is evaluated. Molecular dynamic simulation indicates that the binding mode of hemin to G-quadruplex DNA is end-stacking, which is consistent with absorption spectroscopy. Based on fluorescence spectroscopy, hemin ejects thiazole orange from bases of four-strand DNA. Circular dichroism spectra showed that no alteration occurs in this type of DNA structure.

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Graphical Abstract Peroxidase DNAzyme is formed by hemin and G-quadruplex DNA.

     

Amplified Chemiluminescence Detection of DNA by Strand-Displacement Polymerization Target Recycling and G-quadruplexes/Hemin DNAzyme Catalysis

A new strategy based on strand-displacement polymerization target recycling and G-quadruplex DNAzyme catalysis was developed for amplified chemiluminescence detection of DNA. The amplified detection was achieved by using the system consisted of hairpin DNA probe, G-rich DNA, primer, and polymerase Klenow fragment exo^?. When the target DNA was introduced the system, the hairpin structure of DNA probe was opened by the hybridizing of target DNA with its complementary sequence, the primer hybridized then with DNA probe and initiated polymerase-aided strand-displacement polymerization reaction, resulting in the release of target DNA and G-rich DNA. The released target DNA again hybridizes with another DNA probe to trigger the next polymerase-aided strand-displacement polymerization, generating large numbers of G-rich DNA. The G-rich DNA assembles with hemin to form the G-quadruplexes/hemin DNAzyme, which can catalyze oxidation of luminol by H₂O₂, generating chemiluminescence. This unique amplifying strategy gives a detection limit down to 2.5 pM, which is at least two to three orders of magnitude lower than that of unamplified DNA detection methods.     

Hemoglobin and hemin induce DNA damage in human colon tumor cells HT29 clone 19A and in primary human colonocytes

Epidemiological findings have indicated that red meat increases the likelihood of colorectal cancer. Aim of this study was to investigate whether hemoglobin, or its prosthetic group heme, in red meat, is a genotoxic risk factor for cancer. Human colon tumor cells (HT29 clone 19A) and primary colonocytes were incubated with hemoglobin/hemin and DNA damage was investigated using the comet assay. Cell number, membrane damage, and metabolic activity were measured as parameters of cytotoxicity in both cell types. Effects on cell growth were determined using HT29 clone 19A cells. HT29 clone 19A cells were also used to explore possible pro-oxidative effects of hydrogen peroxide (H2O2) and antigenotoxic effects of the radical scavenger dimethyl sulfoxide (DMSO). Additionally we determined in HT29 clone 19A cells intracellular iron levels after incubation with hemoglobin/hemin. We found that hemoglobin increased DNA damage in primary cells (> or =10 microM) and in HT29 clone 19A cells (> or =250 microM). Hemin was genotoxic in both cell types (500-1000 microM) with concomitant cytotoxicity, detected as membrane damage. In both cell types, hemoglobin and hemin (> or =100 microM) impaired metabolic activity. The growth of HT29 clone 19A cells was reduced by 50 microM hemoglobin and 10 microM hemin, indicating cytotoxicity at genotoxic concentrations. Hemoglobin or hemin did not enhance the genotoxic activity of H2O2 in HT29 clone 19A cells. On the contrary, DMSO reduced the genotoxicity of hemoglobin, which indicated that free radicals were scavenged by DMSO. Intracellular iron increased in hemoglobin/hemin treated HT29 clone 19A cells, reflecting a 40-50% iron uptake for each compound. In conclusion, our studies show that hemoglobin is genotoxic in human colon cells, and that this is associated with free radical mechanisms and with cytotoxicity, especially for hemin. Thus, hemoglobin/hemin, whether available from red meat or from bowel bleeding, may pose genotoxic and cytotoxic risks to human colon cells, both of which contribute to initiation and progression of colorectal carcinogenesis.     

Hemin-stimulated docking of cytochrome c to a hemin-DNA aptamer complex

DNA aptamers were selected for their ability to bind simultaneously to the protein cytochrome c and to the metalloporphyrin hemin. Such aptamers each contained a conserved guanine-rich core, analogous to sequences shown previously to form a hemin-binding site when folded. The detailed study of CH6A, a deletion mutant of one clone, indicated that in the presence of hemin the guanine-rich core of the aptamer folded to form a guanine quadruplex. Both hemin and potassium ions were required for this folding. The binding of fully oxidized cytochrome c to this DNA-hemin complex resulted in an absorbance difference spectrum in the Soret region, which could be used as an indicator of binding behavior. It was found that cytochrome c bound more tightly to the folded CH6A DNA-hemin complex than to the folded CH6A DNA alone. A single hemin molecule and a single cytochrome c bound to each molecule of folded CH6A. Footprinting experiments showed the binding site of the cytochrome c to be a partial duplex element of the aptamer, immediately flanking its guanine-rich hemin-binding site. The order of addition of hemin and cytochrome c appeared not to affect either the formation rate or the structure of the final ternary complex. The ternary complex represents the docking of a nucleic acid-heme complex to cytochrome c (a protein-heme complex). Future experiments will focus on investigating the optimal electron-transfer path between the two iron centers through intervening protein and DNA.     

A cell free system from HeLa cells active in initiation of protein synthesis.

A cell free system programmed by endogenous mRNA and active in initiation of protein synthesis has been obtained from HeLa cells by adding 25-100 muM hemin to the medium used to homogenize the cells. Hemin stabilizes the initiation activity of the extract, which otherwise decays rapidly even at 0 degrees C. The role of hemin in promoting initiation has been examined by fractionating the extracts into ribosomes and postribosomal supernatant (S150). An extract prepared without hemin or the S150 obtained from this extract prepared without hemin or the S150 obtained from this extract inhibits protein synthesis of the extract containing hemin by about 30%. The ribosomes prepared from extracts containing hemin are active in initiation of protein synthesis, whereas the ribosomes obtained from the extracts prepared without hemin show little or no initiation. These results have suggested that addition of hemin prevents the formation of an inhibitor of initiation in the S150 and at the same time protects from inactivation an initiation factor associated with ribosomes or ribosomal subunits. Addition of 2 mM GTP to HeLa extracts stabilizes the initiation activity, though to a smaller degree than hemin. The effects of hemin and GTP are not additive, suggesting that they may act on the same target molecule, though possibly by different mechanisms. The mechanism of action of GTP is discussed in view of similar observations made in the rabbit reticulocyte cell free system.     

Biogenesis of embryonic chick liver delta-aminolevulinate synthase: regulation of the level of mRNA by hemin

The effects of hemin on the concentration of the mRNA for delta-aminolevulinate synthase (ALA synthase) and on the association of the messenger with polysomes were investigated in primary cultures of embryonic chick hepatocytes incubated with allylisopropylacetamide (AIA). A synthetic 24-mer DNA complementary to ALA synthase mRNA was used to determine by solution hybridization the effects of AIA and of AIA plus hemin on the ALA synthase-specific RNA sequences in the cells. The results indicated that ALA synthase mRNA concentrations increased significantly in hepatocytes incubated for 5 h with AIA (0.075 mg/ml), and that hemin in the medium (2 or 10 microM) blocked the increase in the messenger. When delta-aminolevulinic acid (ALA) and FeCl3 were added into the culture medium (1 mM and 5 microM, respectively), the increase in ALA synthase mRNA brought on by AIA was also inhibited. Neither ALA nor FeCl3, when individually added to the cultures, was as effective as the combination of the two. The results with ALA + FeCl3 suggested that stimulation of intracellular production of heme was also effective in blocking the increase in ALA synthase mRNA caused by AIA. Finally, the distributions of ALA synthase mRNA were compared in polysomes isolated from hepatocytes which had been incubated with AIA for 5 h in the presence and absence of 10 microM hemin in the medium. Although a drop was detected in the concentration of ALA synthase mRNA in polysomes from hepatocytes incubated with hemin for 30 min, the decrease was explained by the effect of hemin on the mRNA concentration in the cells.     

On the chemical nature of DNA and RNA modification by a hemin model system.

In order to model the interaction of hemin with DNA and other polynucleotides, we have studied the degradation of DNA, RNA, and polynucleotides of defined structure by [meso-tetrakis(N-methyl-4-pyridyl)porphinato]manganese(III) (MnTMPP) + KHSO5. The activated porphyrin was shown to release adenine, thymine, and cytosine from DNA; RNA degradation afforded adenine, uracil, and cytosine. The same products were obtained from single- and double-stranded DNA oligonucleotides of defined sequence, and also from single-stranded DNA and RNA homopolymers. The overall yield of bases from the dode-canucleotide d(CGCT3A3GCG) was equal to 14% of the nucleotides present initially, indicating that each porphyrin catalyzed the release of approximately 4 bases. Although no guanine was detected as a product from any of the substrates studied, the ability of MnTMPP + KHSO5 to degrade guanine nucleotides was verified by the destruction of pGp, and by the appearance of bands corresponding to guanosine cleavage following treatment of 32P end labeled DNA restriction fragments with activated MnTMPP. Inspection of a number of sites of MnTMPP-promoted cleavage indicated that the process was sequence-selective, occurring primarily at G residues that were part of 5'-TG-3' or 5'-AG-3' sequences, or at T residues. Also formed in much greater abundance were alkali-labile lesions; these were formed largely at guanosine residues. Also studied was the degradation of a 47-nucleotide RNA molecule containing two hairpins. Degradation of the 5'-32P end labeled RNA substrate afforded no distinct, individual bands, suggesting that multiple modes of degradation may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibacterial effect of peptide conjugates with a quaternized chitosan derivative and its estimation by the method of atomic force microscopy

We obtained a number of conjugates based on a quaternized chitosan derivative and antimicrobial peptides (melittin and warnerin) crosslinked by microbial transglutaminase. We determined the optimal conditions for the synthesis (30 minutes, with a mole ratio of peptides and chitosan derivative of 1.4: 100) and studied the antibacterial properties of obtained conjugates. The antibacterial effect of the conjugates was found to be greater than that of their components. The antibacterial activity of the conjugates was determined by the double-dilution method and by atomic force microscopy.     

Identification and cloning of a hemin storage locus involved in the pigmentation phenotype of Yersinia pestis.

The temperature-dependent absorption of sufficient exogenous hemin or Congo red to form pigmented colonies of Yersinia pestis has been termed the pigmentation phenotype (Pgm+). Spontaneous mutation to a Pgm- phenotype results in the loss of a number of divergent physiological characteristics, including the ability to store hemin and to bind Congo red at 26 degrees C. In this study, we generated and isolated transposon insertion mutants that are hemin storage negative (Hms-) and therefore unable to form pigmented colonies. These mutations are due to single mini-kan insertions within a 19.5-kilobase (kb) SalI fragment of chromosomal DNA. Restriction site analysis of eight mutants identified a minimum of six potentially different insertion sites spanning an approximately 10-kb hemin storage (hms) locus. The 19.5-kb SalI fragment (containing approximately 18 kb of Y. pestis DNA and the mini-kan insert) was cloned from one of these mutants, KIM6-2012. By using this cloned fragment as a DNA probe, the mechanism of spontaneous mutation to a Pgm- phenotype was identified as a massive deletion event. The deletion spans at least 18 kb of genomic DNA in spontaneous Pgm- mutants from nine separate strains of Y. pestis. DNA adjacent to the mini-kan insert was used to identify a clone containing a wild-type hms locus. A spontaneous Pgm- mutant of Y pestis KIM containing this clone exhibits an Hms+ phenotype. The hms::mini-kan mutations and cloned wild-type hms locus generated in this study will greatly aid in identifying the function of hemin storage in Y. pestis.     

In vitro selection of hemin-binding catalytic RNA

Catalytic RNAs with peroxidase activity were obtained by the in vitro selection of RNA aptamer-binding hemin. One of the RNA aptamers selected showed binding affinity to hemin with a dissociation constant of 0.8 μM and exhibited high peroxidase activity by forming a complex with hemin. The catalytic efficiency of the RNA–hemin complex was 10-fold higher than that of hemin alone.     

Exploring the Use of a Guanine-Rich Catalytic DNA for Sulfoxide Preparation

A guanine-rich DNA oligonucleotide complexed with hemin was used to catalyze controlled oxygen transfer reactions to different sulfides for sulfoxide preparation in the presence of H₂O₂. Comparable activities were obtained when using fully modified L-DNA. In addition, oligonucleotide immobilization led to an active catalyst which could be successfully recovered and reused without loss of activity.