Self-association of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) and promotion by metal ions

The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP4-) has been measured in D2O at 27 degrees C to elucidate the self-association. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K = 1.9 +/- 0.2 M-1, is only slightly larger than the value for ATP4-, K = 1.3 +/- 0.2 M-1. The self-stacking tendency of epsilon-ATP4- is promoted by a factor of about 4 by (1:1) coordination of Mg2+ to the phosphate moieties, which probably links these together and also neutralizes part of the negative charge; Zn2+ is only about half as effective as Mg2+ in promoting the self-association. This result contrasts with the self-stacking properties of Mg(ATP)2- and Zn(ATP)2-, Zn2+ being considerably more effective in a 1:1 ATP system. It is assumed that due to the enhanced affinity of the N-6/N-7 site of the epsilon-adenine moiety towards Zn2+ repulsion of the bases occurs resulting thus in a lower stacking tendency; in addition, the simple isodesmic model is no longer applicable to the Zn(epsilon-ATP)2- system: to explain the experimental data, the formation of an intermolecular metal ion bridge in the dimeric stacks is proposed. The experimental conditions required for studies of the properties of monomeric epsilon-ATP systems are described. Care should be exercised in employing epsilon-ATP as a probe for ATP.     

Characterization of the metal ion-binding domains from rat alpha- and beta-parvalbumins

We have examined the metal ion-binding domains from rat alpha and beta parvalbumin. We find that the CD-EF fragments differ markedly in their tendency to self-associate. Whereas Ca(2+)-free alpha CD-EF is monomeric, the Ca(2+)-free beta peptide dimerizes weakly (K(2) = 2400 +/- 200 M(-1)). In buffer containing 1.0 mM Ca(2+), the apparent dimerization constant for beta CD-EF (191,000 +/- 29,000 M(-1)) is more than 50 times that of alpha (3400 +/- 200 M(-1)). Alpha CD-EF binds two Ca(2+) with positive cooperativity. Titration calorimetry data afford binding constants of 3.7(0.1) x 10(3) M(-1) and 8.6(0.2) x 10(4) M(-1). Beta CD-EF also binds two Ca(2+) cooperatively but with lower affinity. Equilibrium dialysis yields Adair constants of 4.2(0.1) x 10(3) and 6.1(0.2) x 10(3) M(-1). Significantly, the difference in Ca(2+) affinity is substantially smaller than that observed for the full-length proteins-suggesting that the AB domain can modulate divalent ion affinity. Analysis of beta calorimetry data requires explicit consideration of the self-association behavior. Data collected at low CD-EF concentration are consistent with preferential occupation of the EF site, dimerization of singly bound monomers, and cooperative filling of the CD sites. At higher concentrations, apo-protein dimerization can apparently precede cooperative occupation of the EF sites. In the presence of Ca(2+), alpha CD-EF exhibits higher thermal stability, consistent with its higher Ca(2+) affinity. However, the beta melting temperature shows greater concentration dependence, consistent with its greater tendency to dimerize. Neither fragment exhibits a sigmoidal melting curve in the Ca(2+)-free state, suggesting that the apo-peptides are disordered.     

Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a "ping-pong" scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants (k(cat)) range from 0.1 to 1600s(-1), and bimolecular rate constants (k(cat)/K(m)) range from 10(3) to 10(8)M(-1)s(-1). Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an "outer-sphere" electron transfer model, taking into account high potential of FMN semiquinone/FMNH(-) couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1(')-dibenzyl-4,4(')-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E(1)(7)). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E(1)(7) or hydride-transfer potential (E(7)(Q/QH(-))). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.     

Frontal gel chromatographic analysis of the interaction of a protein with self-associating ligands: aberrant saturation in the binding of flavins to bovine serum albumin

Frontal gel chromatography is an accurate method to obtain the total free ligand concentration of a protein-ligand mixture in which ligands self-associate. The average number of bound ligands per protein molecule is obtained as a function of the total free ligand concentration. The method was applied to the interaction of bovine serum albumin with self-associating flavins. The binding curves for FMN and FAD leveled off at about 0.7 and 0.5, respectively. These data were simulated well by a binding model where flavins undergo isodesmic indefinite self-association and the monomer alone binds to a single binding site of albumin. The isodesmic association constants of FMN and FAD were (1.7 +/- 0.1) x 10(2) and (2.2 +/- 0.3) x 10(2) M(-1), respectively. The binding constants of the monomer of FMN and FAD were (7.6 +/- 0.2) x 10(2) and (3.5 +/- 0.2) x 10(2) M(-1), respectively. FMN competitively inhibited the binding of FAD to albumin. The affinity to flavins was in the following order at pH 5.8: lumiflavin, FMN, riboflavin, and FAD. The SH modification and the binding of palmitate did not affect the FMN binding to bovine albumin at pH 5.8. As pH increased from 5.8 to 9.0, the affinity to FMN of bovine albumin decreased 3-fold, whereas that of human albumin increased about 80-fold. The present study clearly showed how isodesmic self-association of a ligand can cause apparent saturation of the interaction of a protein with the ligand at levels lower than 1.     

Analysis of proton release in oxygen binding by hemoglobin: implications for the cooperative mechanism

The relationship in hemoglobin between cooperativity (dependence of the Hill constant on pH0 and the Bohr effect (dependence of the mean oxygen affinity on pH) can be described by a statistical thermodynamic model [Szabo, A., & Karplus, M. (1972) J. Mol. Biol. 72, 163-197; Lee, A., & Karplus, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7055-759]. In this model, salt bridges and other interactions serve to couple tertiary and quaternary structural changes. To test and refine the model, it is applied to the analysis of the pH dependence of the tetramer Adair constants corrected for statistical factors (K4i', i = 1-4). Attention is focused on the proton release of the first (delta H1+ = alpha log K41'/alpha pH) and last (delta H4+ = alpha log K44'/alpha pH) oxygenation steps, where K4i' are the Adair constants corrected for statistical factors. Measurements of delta H1+ and delta H4+ under carefully controlled conditions are reported, and good agreement between the model calculation and these experimental results is obtained. The salt bridges are found to be partially coupled to the ligation state in the deoxy quaternary structure; it is shown that a Monod-Wyman-Changeux-type model, in which the salt bridges are coupled only to quaternary structural change, is inconsistent with the data for delta H1. The significance of the present analysis for an evaluation of the Perutz mechanism [Perutz, M.F. (1970) Nature (London) 228, 726-734, 734-739] and other models for hemoglobin cooperativity is discussed.     

Cooperative interaction of histone H1 with DNA.

The cooperative binding of histone H1 with DNA was studied using a fluorescently labelled histone H1. The titration data were analysed in terms of the large ligand model. The stoichiometric number, n = 65 +/- 10 bases/H1, was independent of NaCl concentration (0.02 - 0.35 M). The nucleation and the cooperative binding constants, K' and K, and the cooperativity parameter q were sensitive to salt concentration; K = 3.6 +/- 0.8 X 10(7) M-1 and q = 1.1 +/- 0.4 X 10(3) at 0.2 M NaCl. The dependence of K' on NaCl concentration revealed that 6 Na+ ions were released from DNA upon complex formation. An extrapolation of K' to 1M NaCl yielded a small value, K' = 5 +/- 2 M-1. Thus the binding of H1 is essentially electrostatic, being compatible with its independence of temperature. A calculation of K' based on the counterion release reproduced the salt concentration dependence of K'. Therefore, the binding of H1 is of an electrostatic territorial type. Thus, H1 may move along the DNA chain to a certain extent, when both salt concentration and the degree of saturation are sufficiently low. The condition is so restricted that the sliding would not play an important role in vivo. It was concluded from the DNA concentration independent binding isotherm that H1 can cooperatively bind onto a single DNA molecule. A simple power law dependence of the cooperativity parameter q upon NaCl concentration was found; q oc[NaCl]h with h = 0.72, though the physical basis of this dependence remains unknown.     

Kinetics and mechanism for the conformational transition in p-guanidinobenzoate bovine trypsinogen induced by the isoleucine-valine dipeptide

The interaction between p-guanidinobenzoate-trypsinogen and the isoleucine-valine dipeptide has been investigated by temperature-jump relaxation spectrometry. Using the absorbance at 281 nm the concentration dependence of the relaxation parameters is consistent with the conventional induced-fit model: rapid ligand binding coupled to a slower intramolecular change; some alternative mechanisms can be excluded. At 296 K, 0.1 M Tris HCl, pH = 7.4, the dissociation equilibrium constant for the overall process is K = 5.1(+/- 0.2) X 10(-5) M; for the binding step K1 = 2.3(+/- 0.3) X 10(-3) M and the rate constants for the structural change are k2 = 26(+/-6)s-1 and k-2 = 0.61(+/- 0.04)s-1; the overall dissociation reaction enthalpy is delta H0 = 26(+/-6)KJmol-1 and the reactiom entropy is delta S0 = 4(+/- 20) kJ-1 mol-1. In combination with CD and X-ray crystallographic data, the results of this study suggest that the binding of the dipeptide to a trypsinogen-like, partially disordered conformation induces a transition to a trypsin-like highly ordered structure.     

Gelatin-alpha olefin sulfonate interactions studied by dynamic light scattering

Dynamic light scattering (DLS) measurements were performed to study the binding of anionic surfactant alpha olefin sulfonate (AOS) to gelatin chains at various NaCl concentrations at 30 degrees C in aqueous sodium phosphate buffer (pH = 6.8) solutions. The surfactant concentration was varied from 0 to 80 mM and the NaCl concentrations chosen were 0.025, 0.05, and 0.1 M. AOS exhibited electrostatic binding to the positively charged sites of the polypeptide chain resulting in considerable reduction in its hydrodynamic radius up to critical micellar concentration (cmc = 8 mM for no salt, 0.01 and 0.025 M, and 5 mM for 0.05 M and 2 mM for 0.1 M solutions). The correlation function revealed the presence of two types of structures above cmc; namely the micelles of AOS and gelatin-AOS micelle complexes. The micellar radii (Rm), the effective gelatin-surfactant complex radii (Rc), have been determined as a function of salt concentration. No critical aggregation concentration (cac) was observed. The inter-gelatin-surfactant complex (kD1) and inter-micellar interactions (kD2), were determined by fitting the concentration dependence of Rm and Rc to a virial expansion in reduced concentration (c - cmc), which are compared. While kD1 showed strong ionic strength dependence, kD2 remained invariant of the same. The protein to surfactant binding ratio was found to be smaller than normal. Results have been discussed within the framework of the necklace-bead model of polymer-surfactant interactions.     

Electron donors and inhibitors of nitrate reductase from Cyanidium caldarium.

Studies on nitrate reductase (NAD(P)H:nitrate oxidoreductases EC 1.6.6.2) of Cyanidium caldarium revealed that the enzyme is inhibited by excess of electron donor, NADPH, reduced benzylviologen and FMN. Also dithionite, used to reduce benzylviologen and FMN, inactivates nitrate reductase: however, FMN at an optimal concentration and nitrate, added before the dithionite, protect the enzyme against this inactivation. Cyanide, cyanate and carbamyl phosphate inhibit the enzyme competitively with respect to nitrate, and Ki values are reported. Organic mercurials, 0.1 mM, act preferentially on NADPH activity, whereas Ag+ and Hg-2+ at the same concentration inactivate 80--90% of the benzylviologen and FMN activities. ADP is very poor inhibitor. Urea 4 M in 2 h destroys 90% of the NADPH activity and only 30% of the benzylviologen and FMN activities. The apparent Km values for NADPH, benzylviologen, FMN and nitrate have been determined.     

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.     

Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex.

The kinetics of the reaction of trypsin with alpha 2M were examined under pseudo-first-order conditions with excess inhibitor. Initial studies indicated that the fluorescent dye TNS is a suitable probe for monitoring the reaction over a wide concentration range of reactants. Titration experiments showed that the conformational changes associated with the binding of trypsin to alpha 2M result in an increased affinity of the inhibitor for TNS. Two distinct phases were observed when this dye was used to monitor the progress of the reaction. Approximately half of the fluorescence signal was generated during a rapid phase, with the remainder generated during a second, slower phase. The observed pseudo-first-order rate constant of the first phase varied linearly with the concentration of alpha 2M up to the highest concentration of inhibitor used, whereas the rate constant of the second phase was independent of alpha 2M concentration. The data fit a mechanism in which the association of trypsin with alpha 2M occurs in two consecutive, essentially irreversible steps, both leading to alterations in TNS fluorescence. The initial association occurs with a second-order rate constant of (1.0 +/- 0.1) X 10(7) M-1 s-1 and is followed by a slower, intramolecular conformational rearrangement of the initial complex with a rate constant of 1.4 +/- 0.2 s-1. The data are consistent with a previously proposed model for the reaction of proteinases with alpha 2M [Larsson et al. (1989) Biochemistry 28, 7636-7643].2+ this model, once an initial 1:1 alpha 2M-proteinase     

No cofactor effect on equilibrium unfolding of Desulfovibrio desulfuricans flavodoxin

Flavodoxins are proteins with an alpha/beta doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin (K(D)=0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (DeltaG=20(+/-2) and 19(+/-1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high (K(D)=0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.     

Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.     

The effect of temperature on FMN absorption spectra in rigid poly(vinyl alcohol) matrices

Electronic absorption spectra of flavomononucleotide (FMN) in poly(vinyl alcohol) films (PVA) were measured over the concentrations ranging from 6.9 x 10(-4) to 6.8 x 10(-1) M and temperatures from 263 to 338 K.The FMN absorption spectra measurements performed at room temperature have shown two ranges of different changes as a function of dye concentration. For concentrations c<10(-1) M (range I) the spectra exhibited regular changes showing an isosbestic point, which evidences the equilibrium between monomers and dimers. However, for range II (c>1.05 x 10(-1) M) the FMN absorption spectra occurred to be almost independent of concentration and they nearly overlapped with the dimer spectrum (within the error limit).Temperature measurements have shown that the FMN absorption spectra in PVA are stable over a wide temperature range.The mean distances between FMN molecules in PVA films are calculated. For maximal concentrations (from the range II), they are below 13.1 A, whereas the mean dimensions of FMN monomers and dimers are 15.8 and 21.1 A, respectively, which indicates that the orientation of dimers and monomers in the PVA film cannot be random at high concentrations. Molecules are partly ordered, adopting approximately parallel orientation, which is in agreement with the calculations of dimer structure by molecular modelling method (MMM).     

Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry

Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.     

alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.     

Kinetics and mechanism of electron transfer between meso-tetra sulphonated phenyl porphyrin iron(III)-apomyoglobin and Fe(EDTA)2- complexes

The kinetics of electron transfer between Fe(EDTA)2- and meso-tetra sulphonated phenyl porphyrin iron(III)-apomyoglobin have been studied by applying stopped-flow mixing and monitoring photometric changes at soret band (429 nm). The studies were carried out at pH's 6, 6.5, 7, 7.5, and 8 and at temperature between 10 and 40 degrees C. The mechanism proposed on the basis of the dependence of kobsd on Fe(EDTA)2- concentrations at various pH's, followed the rate equation: kobsd = ka[H+] + Kakb/[H+] + Ka.[Fe(EDTA)2-] The values of rate parameters calculated using a weighted non-linear least-squares analysis were: ka, 528 +/- 2 sec-1; kb, 25 +/- 1 sec-1; and Ka, 2.0 +/- 0.1 microM at 25 degrees C and 0.5 M sodium phosphate, and those of thermodynamic parameters calculated by the Eyring equation were: delta H*, 8.1 +/- 0.3 kcal mole-1 and delta S*, -23.4 +/- 1.1 eu at pH 7 and 0.5 M sodium phosphate.     

Sequence specificity modification of double-stranded DNA with an alkylating derivative of oligodeoxyribonucleotide pT(CT)6

It is shown that in slightly acidic solution (pH approximately 5.3) reagent CIRCH2NHpT(CT)6 (RCl = -C6H4-N(CH3)CH2CH2Cl) modifies a double-stranded DNA fragment (120 b. p.) containing A(GA)6.T(CT)6 sequence at a single nucleotide residue, viz. G29 located near to this sequence in the DNA chain. The location of this modification point suggests formation of a triple-stranded reactive complex with parallel orientation of the pyrimidine oligonucleotide moiety of the reagent and pyrine sequence of the target DNA. Analysing the modification extent dependence of the reagent concentration the association constant Kx between the reagent and DNA was calculated (Kx = (0.95 +/- 0.03).10(5) M-1, 25 degrees C, pH = 5.3, [NaCl] = 0.1 M). The modification by the reagent ClRCH2NHpT(m5CT)6 has the same quantitative characteristics as in the case of ClRCH2NHpT(CT)6.     

Reaction of proteinases with alpha 2-macroglobulin: evidence for alternate reaction pathways in the inhibition of trypsin

Titration experiments were employed to measure the binding stoichiometry of alpha 2M for trypsin at high and low concentrations of reactants. These titration experiments were performed by measuring the SBTI-resistant trypsin activity and by direct binding measurements using 125I-labeled trypsin. The binding stoichiometry displayed a marked dependence upon protein concentration. At high alpha 2M concentrations (micromolar), 2 mol of trypsin are bound/mol of inhibitor. However, at low alpha 2M concentrations (e.g., 0.5 nM), only 1.3 mol of trypsin were bound/mol of inhibitor. Sequential additions of subsaturating amounts of trypsin to a single aliquot of alpha 2M also resulted in a reduction in the final binding ratio. A model has been formulated to account for these observations. A key element of this model is the observation that purified 1:1 alpha 2M-proteinase complexes are not capable of binding a full mole of additional proteinase [Strickland et al. (1988) Biochemistry 27, 1458-1466]. The model predicts that once the 1:1 alpha 2M-proteinase complex forms, this species undergoes a time-dependent conformational rearrangement to yield a complex with greatly reduced proteinase binding ability. According to this model, the ability of alpha 2M to bind 2 mol of proteinase depends upon the association rate of the second enzyme molecule with the binary (1:1) complex, the enzyme concentration, and the rate of the conformational alteration that occurs once the initial complex forms. Modeling experiments suggest that the magnitude of the rate constant for this conformational change is in the order of 1-2 s-1.     

On the metal-ion coordinating properties of the 5'-monophosphates of 1, N6-ethenoadenosine (epsilon-AMP), adenosine and uridine. Comparison of the macrochelate formation in the complexes of epsilon-AMP, AMP, ADP and ATP

The concentration dependence of the chemical shifts of the protons H-2, H-8, H-10, H-11, and H-1' of 1,N6-ethenoadenosine 5'-monophosphate (epsilon-AMP2-) has been measured. The results are consistent with the isodesmic model of indefinite noncooperative stacking; the association constant, K = 2.5 +/- 0.3 M-1, is within experimental error identical to the value determined earlier for AMP2-,K = 2.1 +/- 0.4 M-1. The conditions for the potentiometric pH titrations, used to determine the acidity constants of H2(epsilon-AMP), H2(AMP), and H(UMP)- and the stability constants of the metal ion (M2+) complexes of the corresponding nucleoside 5'-monophosphates (NMP), were chosen so that the ligands were present in the monomeric form. The stabilities of Mg(epsilon-AMP) and Mg(AMP) are similar; however, the stabilities of the Mn2+, Cu2+ and Zn2+ complexes of epsilon-AMP2- are much larger (in the case of Cu2+ by a factor of 700) than those of AMP2-. This is due to the much larger metal ion affinity of the epsilon-adenosine moiety compared to that of the parent adenosine residue. As the uridine moiety does not participate in complex formation, the stability constants of M(UMP) have been used to evaluate the extent of macrochelation (i.e. the simultaneous coordination of M2+ to the base moiety and the phosphate group) in the epsilon-AMP and AMP complexes: the concentration of the macrochelated isomer is considerably larger for M(epsilon-AMP) than for M(AMP). A comparison with previous results for the complexes with ADP3- and ATP4- indicates the order, M(AMP)cl less than M(ADP)-cl greater than M(ATP)2-cl for the tendency to form macrochelates (cl). Due to the relatively high affinity of the epsilon-adenosine moiety towards Mn2+, Cu2+ and Zn2+, the phosphate-monoprotonated complexes M(H . epsilon-AMP)+ also become important; the corresponding complexes play only a minor role in the M2+/AMP systems. Intramolecular aromatic-ring stacking occurs in the ternary Cu(2,2'-bipyridyl)(NMP) complexes: about 80% of Cu(Bpy)(AMP) and Cu(Bpy)(epsilon-AMP) exist as the stacked isomer in aqueous solution; for the former system it has been shown in a previous X-ray study that the intramolecular ligand-ligand interaction occurs also in the solid state [Aoki, K. (1978) J. Am. Chem. Soc. 100, 7106]. Overall, the results emphasize that great care should be exercised in drawing conclusions based on studies of metal-ion-containing enzymic systems in which the natural adenine nucleotide cofactors have been replaced by the corresponding 1,N6-etheno derivatives.