DNA tris-intercalation: first acridine trimer with DNA affinity in the range of DNA regulatory proteins. Kinetic studies

A trimer made up of three acridine chromophores linked by a positively charged poly(aminoalkyl) chain was synthesized as a potential tris-intercalating agent. The length of the linking chain was selected to allow intercalation of each chromophore according to the excluded site model. 1H NMR studies have shown that, at 5 mM sodium, pH 5, the acridine trimer occurred under a folded conformation stabilized by stacking interactions between the three aromatic rings. DNA tris-intercalation of the dye at a low dye/base pair ratio was shown by measurements of both the unwinding of PM2 DNA and the lengthening of sonicated rodlike DNA. The trimer exhibits a high DNA affinity for poly[d(A-T)] (Kapp = 8 X 10(8) M-1, 1 M sodium) as shown by competition experiments with ethidium dimer. Kinetic studies of both the association with poly[d(A-T)] and the exchange between poly[d(A-T)] and sonicated calf thymus DNA have been performed as a function of the ionic strength. In 0.3 M sodium the on-rate constant (k1 = 2.6 X 10(7) M-1 s-1) is similar to that reported for other monoacridines or bis(acridines), whereas the off-rate constant is much smaller (k-1 = 1.2 X 10(-4) s-1), leading to an equilibrium binding constant as large as Kapp = 2.2 X 10(11) M-1. A plot of log (k1/k-1) as a function of log [Na+] yielded a straight line whose slope shows that 5.7 ion pairs (out of 7 potential) are formed upon the interaction with DNA. From this linear relationship a Kapp value of 10(14) M-1 in 0.1 M sodium can be estimated. Such a value reaches and even goes beyond that of some DNA regulatory proteins. This acridine trimer appears to be the first synthetic ligand with such a high DNA affinity.     

The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin

Analysis of two recombinant variants of chicken striated muscle alpha-tropomyosin has shown that the structure of the amino terminus is crucial for most aspects of tropomyosin function: affinity to actin, promotion of binding to actin by troponin, and regulation of the actomyosin MgATPase. Initial characterization of variants expressed and isolated from Escherichia coli has been published (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Fusion tropomyosin contains 80 amino acids of a nonstructural influenza virus protein (NS1) on the amino terminus. Nonfusion tropomyosin is a variant because the amino-terminal methionine is not acetylated (unacetylated tropomyosin). The affinity of tropomyosin labeled at Cys190 with N-[14C]ethylmaleimide for actin was measured by cosedimentation in a Beckman Airfuge. Fusion tropomyosin binds to actin with an affinity slightly greater than that of chicken striated muscle alpha-tropomyosin (Kapp = 1-2 X 10(7) versus 0.5-1 X 10(7) M-1) and more strongly than unacetylated tropomyosin (Kapp = 3 X 10(5) M-1). Both variants bind cooperatively to actin. Troponin increases the affinity of unacetylated tropomyosin for actin (+Ca2+, Kapp = 6 X 10(6) M-1; +EGTA, Kapp = 2 X 10(7) M-1), but the affinity is still lower than that of muscle tropomyosin for actin in the presence of troponin (Kapp much greater than 10(8) M-1). Troponin has no effect on the affinity of fusion tropomyosin for actin indicating that binding of troponin T to the over-lap region of the adjacent tropomyosin, presumably sterically prevented by the fusion peptide in fusion tropomyosin, is required for troponin to promote the binding of tropomyosin to actin. The role of troponin T in regulation and the mechanisms of cooperative binding of tropomyosin to actin have been discussed in relation to this work.     

The affinity of DNA-microtubule protein complexes and their disruption by tubulin binding drugs.

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar Kapp values in the range of 2.1 x 10(7) M-1 to 2.0 x 10(8) M-1. CEN11 DNA-MTP complexes had by far the highest Kapp value of 2.0 x 10(8) M-1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites-(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher Kapp value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10-100 fold higher drug concentrations to disrupt the CEN11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383-392, 1984; Marx and Denial, Molecular Basis of Cancer 172B, 65-75 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.     

Time-resolved fluorescence of bacteriophage Pf1 DNA-binding protein. Determination of oligonucleotide and polynucleotide binding parameters

The binding of oligonucleotides and polynucleotides to the Pf1 DNA-binding protein was followed by fluorescence spectral shift and lifetime measurements, which gave an anomalous value for the stoichiometry of binding. The anomaly was investigated in detail using fluorescence depolarisation to measure the aggregation during the titration and showed that all the fluorescence parameters are related to the specific aggregation of dimers on ligand binding. At saturation, complexes of the protein with the octanucleotide d(GCGTTGCG) and the hexadecanucleotide (dT)16 have rotational correlation times, phi, of 50 ns and 85 ns, corresponding to protein tetramers and octamers, respectively. In the presence of the tetranucleotide d(CGCA) the protein remains as the native dimer (phi = 19 ns). The titration curves could be analysed in terms of two non-equivalent binding sites, with binding constants K1 and K2. Comparison of K1 values for oligonucleotide binding leads to an estimated (single-site) intrinsic binding constant Kint approximately equal to 3 X 10(4) M-1 and a cooperativity parameter omega approximately equal to 100, in agreement with the apparent binding constant Kapp approximately equal to 3 X 10(6) M-1 for polynucleotides. Binding to the second site on the protein dimer is greatly reduced and cannot be determined accurately. The results suggest that the protein dimers bind cooperatively by lateral association along the DNA and that occupation of only one of the two DNA-binding sites of the protein dimers is sufficient to stabilize the nucleoprotein complexes.     

Structural basis of anthracycline selectivity for unilamellar phosphatidylcholine vesicles: an equilibrium binding study

Fluorescence anisotropy titration was used to determine the equilibrium binding affinities of several anthracycline antitumor antibiotics for sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Eight daunomycin analogues, all differing from the parent by one structural change in the aglycon portion of the molecule, as well as four anthracycline congeners modified in the amino sugar were studied. Double-reciprocal plots were used to determine overall binding affinities (K). It was shown that structural changes in both the aglycon and amino sugar portions of the daunomycin molecule strongly modulated K values for DMPC and DPPC bilayers. For modifications in the aglycon portion of an anthracycline, a correlation between drug hydrophobicity and membrane affinity was observed. The number of binding sites per phospholipid molecule (n) and the apparent association constant (Kapp) where K = nKapp, were determined at several temperatures for adriamycin, daunomycin, and carminomycin. The n values were found to be independent of temperature for fluid-phase DMPC or solid-phase DPPC bilayers. The Kapp values (25 degrees C) ranged from (0.82-4.4) X 10(5) M-1 for DMPC vesicles to (4.4-7.3) X 10(5) M-1 for DPPC vesicles. Although the Kapp values for the three drugs were similar for a particular bilayer, major differences were noted in the values of n and, therefore, in the overall vesicle affinities (nKapp). van't Hoff plots showed that anthracycline binding was exothermic; in all cases but one binding was accompanied by a decrease in entropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of pyronin Y(G) with nucleic acids

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.     

Nucleic acid binding affinity of fd gene 5 protein in the cooperative binding mode

A sensitive ESR method which allows a direct quantitative determination of nucleic acid binding affinities of proteins under physiologically relevant conditions has been applied to the gene 5 protein of bacteriophage fd. This was achieved with two spin-labeled nucleic acids, (ldT, dT)n and (lA,A)n, which served as macro-molecular spin probes in ESR competition experiments. With the two different macromolecular spin probes, it was possible to determine the relative apparent affinity constants, Kapp, over a large affinity domain. In 20 mM Tris X HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton, and 125 mM NaCl, the following affinity relationship was observed: K(dT)napp = 10(3) KfdDNAapp = 2 X 10(4) K(A)napp = 6.6 X 10(4) KrRNAapp = 1.5 X 10(5) KR17RNAapp. Increasing the [NaCl] from 125 to 200 mM caused considerably less tight binding of gene 5 protein to (lA,A)n, and a typical cooperative binding isotherm was observed, whereas at the lower [NaCl] used for the competition experiments, the binding was essentially stoichiometric. A computer fit of the experimental titration data at 200 mM NaCl gave an intrinsic binding constant, Kint, of 1300 M-1 and a cooperativity factor, omega, of 60 (Kint omega = Kapp) for (lA,A)n.     

Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.     

Static and kinetic studies on carp muscle parvalbumins

Fluorescence titration and fluorescence stopped-flow studies were performed on carp muscle parvalbumin components 1, 2, 3, and 5 (the latter three components were modified with a SH-directed fluorescent reagent, dansyl-L-cysteine). Apparent binding constants (Kapp) of Ca2+ to these components decrease in the order of component 2 (Kapp = 2.8 +/- 0.9 X 10(8) M-1) greater than component 1 (Kapp = 1.25 +/- 0.25 X 10(8) M-1) greater than component 3 = component 5 (Kapp = 4.0 +/- 0.5 X 10(7) M-1) in 30 mM KCl, 50 mM Na-cacodylate-HCl, pH 7.0 at 20 degrees C. The rate constant of the conformational change of parvalbumin induced by Ca2+ binding or removal decreases in the order of component 2 greater than component 1 greater than component 5 greater than or equal to component 3; that is, component 2 undergoes the fastest conformational change and component 3 the slowest in response to the rapid free Ca2+ concentration ([Ca2+]) change in the protein solution. The fluorescence titration curves and [Ca2+]-dependences of the rate constants are analyzed by a simple two-state model, (partially unfolded state) k1 in equilibrium k2 (folded state). It is shown that the equilibrium constant K = k1/k2 depends on the second power of [Ca2+], the rate constant k1 on the first power of [Ca2+] and k2 on the inverse first power of [Ca2+], respectively.     

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.     

Kinetics of carbon monoxide binding to singly reduced human methemoglobin

The kinetics of reaction of singly reduced methemoglobin (HbFe3(3+)Fe2+) with carbon monoxide have been investigated by the pulse radiolysis method. The rate constant for carbon monoxide binding to this form of hemoglobin is 4.1 X 10(6) M-1 S-1 at 24 degrees in our solutions. This value compares with existing values for various forms of hemoglobin ranging from 4 X 10(6) to 6.5 X 10(6) M-1 S-1. Addition of inositol hexaphosphate to the solutions results in a lower rate constant for carbon monoxide binding amounting to 1.1 X 10(5) M-1 S-1.     

Oxidative degradation of cationic metalloporphyrins in the presence of nucleic acids: a way to binding constants?

Mn(III) and Fe(III) complexes of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (M-TMePyP) and related hybrid molecules ("metalloporphyrin-ellipticine") were activated by potassium monopersulfate in the presence of variable calf thymus (CT) DNA and NaCl concentrations. Monitored by visible spectroscopy (Soret band), fast degradation of the free metalloprophyrin was observed while the DNA-bound form appeared protected. This direct quantitation of free versus bound metalloporphyrin ratios allowed determination of binding constants: Mn- and Fe-TMePyP respectively bind to CT DNA (5 mM phosphate buffer, 0.1 M NaCl, pH 7) with K = 3 X 10(4) and 1.2 X 10(4) M-1. Mn-TMePyP showed a greater affinity for poly[d(A-T)] (K = 1.2 X 10(5) M-1) than for poly[d(G-C)] (K = 0.2 X 10(4) M-1). This method allowed us access to the intrinsic DNA affinity of the metalloporphyrin moiety of the hybrid molecules "metalloporphyrin-ellipticine".     

Interactions of retroviral structural proteins with single-stranded nucleic acids

We have studied the interactions of single-stranded polyribonucleotides with murine leukemia virus structural proteins p10, p10' (a p10 variant), and Pr65gag, as well as Rous sarcoma virus (RSV) pp12 (a p10 analog). Two quantitative assays have been used to monitor protein-RNA association: the fluorescence enhancement of polyethenoadenylic acid) poly(epsilon A) upon binding protein, and tryptophan fluorescence quenching upon binding to poly(U). With each assay p10 was shown to bind stoichiometrically to single-stranded RNA, covering a length of nucleic acid chain (occluded site size, n) of about 6 residues. RSV pp12 was also shown to bind to poly(epsilon A), with n = 5 +/- 1. Addition of NaCl to fully titrated MuLV p10-nucleic acid mixtures effected nearly complete restoration of poly(epsilon A) or MuLV p10 fluorescence. Under conditions of 0.06 M NaCl, p10 bound noncooperatively to poly(epsilon A) with an intrinsic association constant, K = 2.3 X 10(6) M-1. K and n determined in this study were shown to relate to Kapp determined by other methods, by the approximation Kapp approximately NK, where N is the number of binding sites along the polynucleotide chain ((nucleotides/chain)/n). Chemical modifications of the p10 cysteine residues did not alter the affinity for poly(epsilon A). The affinity of Pr65gag for poly(epsilon A) appears to be higher than that of p10.     

Interactions of cis- and trans-platinum(II) complexes with dehydrogenase enzymes in the presence of different mono- and polynucleotides: evidence for a ternary complex

The inhibition of several dehydrogenase enzymes by cis- and trans-Pt(NH3)2Cl2 have been measured in the presence of baker yeast ribonucleic acid (RNA), calf thymus and salmon sperm deoxyribonuclic acid (DNA) and several mononucleotides (AMP and ATP). The binding constants for the interaction of the platinum complexes to the nucleotides have been calculated and a comparison of those values to the previously calculated platinum complex-enzyme binding constants strongly suggest that platinum compounds are more tightly bound to the enzymes. The binding of the platinum complexes to most of the enzymes was decreased in the presence of any nucleotide, yet it was observed that when using rabbit muscle (M4) lactate dehydrogenase the mononucleotides reduced the binding to a lesser degree while the polynucleotides actually enhanced the platinum-enzyme interaction. The implications of these interactions are discussed.     

Interaction of cationic porphyrins with DNA: importance of the number and position of the charges and minimum structural requirements for intercalation

Thirty-three porphyrins or metalloporphyrins corresponding to the general formula [meso-[N-methyl-4(or 3 or 2)-pyridiniumyl]n(aryl)4-nporphyrin]M (M = H2, CuII, or ClFeIII), with n = 2-4, have been synthesized and characterized by UV-visible and 1H NMR spectroscopy and mass spectrometry. These porphyrins differ not only in the number (2-4) and position of their cationic charges but also in the steric requirements to reach even temporarily a completely planar geometry. In particular, they contain 0, 1, 2, 3, or 4 meso-aryl substituents not able to rotate. Interaction of these porphyrins or metalloporphyrins with calf thymus DNA has been studied and their apparent affinity binding constants have been determined by use of a competition method with ethidium bromide which was applicable not only for all the free base porphyrins but also for their copper(II) or iron(III) complexes. Whatever their mode of binding may be, their apparent affinity binding constants were relatively high (Kapp between 1.2 x 10(7) and 5 x 10(4) M-1 under our conditions), and a linear decrease of log Kapp with the number of porphyrin charges was observed. Studies of porphyrin-DNA interactions by UV and fluorescence spectroscopy, viscosimetry, and fluorescence energy transfer experiments showed that not only the tetracationic meso-tetrakis[N-methyl-4(or 3)-pyridiniumyl]porphyrins, which both involved four freely rotating meso-aryl groups, but also the corresponding tri- and dicationic porphyrins were able to intercalate into calf thymus DNA. Moreover, the cis dicationic meso-bis(N-methyl-2-pyridiniumyl)diphenylporphyrin, which involved only two freely rotating meso-aryl groups in a cis position, was also able to intercalate. The other meso-(N-methyl-2-pyridiniumyl)n(phenyl)4-nporphyrins, which involved either zero, one, or two trans freely rotating meso-aryl groups, could not intercalate into DNA. These results show that only half of the porphyrin ring is necessary for intercalation to occur.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

Interactions of the high-mobility-group-like Ceratitis capitata C1 proteins with DNA

We have studied the interactions of the high-mobility-group-like proteins (C1a1, C1a2 and C1b) from the fruit fly Ceratitis capitata with DNA. Nitrocellulose filter binding assays, thermal denaturation studies and spectrofluorimetry of the complexes revealed the existence of specific and nonspecific interactions. Thermal denaturation curves showed that the three proteins stabilized the DNA, thus suggesting a preferential binding to double-stranded DNA. The calculation of the thermodynamic parameters of the interactions showed that the nonspecific bindings were characterized by low association constants (Ka) with values ranging from 2.7 X 10(4) M-1 to 2.0 X 10(6) M-1. Also, the cooperativity of these interactions was relatively high (cooperativity factor, w, values ranging over 20-35), and the number of nucleotides involved was low (1-3 base pairs). On the other hand, the existence of specific interactions between C1 proteins and DNA was suggested by two facts: the retention of C. capitata [3H]DNA in nitrocellulose filters was only a low percentage of total input DNA and there was a marked size dependence of the binding (25% retention of a 40-kb DNA and only 3% retention with a DNA of 1 kb). The specific bindings had higher Ka values than the nonspecific ones, and they also were cooperative. Some differences were observed between C1b and the C1a proteins about the way they interact with C. capitata DNA.     

Cleavage of DNA by electrochemically activated MnIII and FeIII complexes of meso-tetrakis (N-methyl-4-pyridiniumyl)porphine

Electrochemical methods were used to activate MnIII and FeIII complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2TMPyP) to cause cleavage of pBR322 DNA and to study their interaction with sonicated calf thymus DNA. Electrochemical reduction of MnIIITMPyP and FeIIITMPyP (at low concentrations) in the presence of O2 was required to activate these complexes. However, FeIIITMPyP at 1 x 10(-6) M produced DNA strand breakage without being electrochemically reduced. At low concentrations, FeIITMPyP was more efficient at cleaving DNA than MnIITMPyP. Reduction of O2 at a platinum electrode also produced some cleavage but to a much smaller extent. The oxidized form of MnIIITMPyP (charge 5+) has higher affinity for sonicated calf thymus (CT) DNA than the reduced form (charge 4+), as determined by the negative shift in E degrees' for the voltammetric wave in the presence of DNA. Both forms of FeIIITMPyP (charge 4+) interact with DNA to about the same extent. Differential pulse voltammetry was used to determine binding constants (K) and binding-site sizes (s) of the interaction of these metalloporphyrins with sonicated CT DNA. The data were analyzed assuming both mobile and static equilibria. MnIIITMPyP binds to DNA (5 mM Tris, 50 mM NaCl, pH 7) with K = 5 (+/- 2) x 10(6) M-1, s = 3 bp (mobile) or K = 3.6 (+/- 0.3) x 10(6) M-1, s = 4 bp (static). FeIIITMPyP at that ionic strength caused DNA precipitation. At higher ionic strength (0.1 M Tris, 0.1 M NaCl, pH 7), FeIIITMPyP associates to DNA with K = 4.4 (+/- 0.2) x 10(4) M-1, s = 5 bp (mobile) or K = 1.9 (+/- 0.1) x 10(4) M-1, s = 6 bp (static).