37 research outputs found
Electron spin resonance studies of the ligand exchange in cupric complexes with diethyldithiocarbamate and diethyldithiophosphate as ligands
The kinetics of ligand substitutions in the cupric complexes where a diethyldithiocarbamate (dtc) is replaced by a diethyldithiophosphate (dtp) has been studied with chloroform as solvent. The relative concentrations of the paramagnetic species produced in the reactions have been determined using electron spin resonance. The reaction studies suggest the presence of the short time equilibria: Cu (dtc) + dtpH ⇋ Cu (dtc) (dtp) + dtcH Cu (dtc) (dtp) + dtpH⇋Cu (dtp) + dtcH where the equilibrium constants have the value, 0.0625 and 0.00143 respectively. The equilibria are disturbed by the reaction: dtcH + dtpH → (C H)2 NH + dtp+ CS with a rate constantk = 0.07 1. mole sec. at 26°C. Analysis of the thermodynamic parameters determined from the study of the kinetics of the reactions at various temperatures shows that dtp group favours more solvent and excess ligand coordination than dtc. This is consistent with the study of "Long time equilibrium" which indicates that Cu (dtp) exists with further two weakly bonded dtpH groups in the axial positions in chloroform solutions
Determination of Bonding Parameters in Cupric Complexes from ESR Linewidth Studies in Liquid Solutions
290-29
Two mechanisms of H+/OH- transport across phospholipid vesicular membrane facilitated by gramicidin A.
Two rate-limiting mechanisms have been proposed to explain the gramicidin channel facilitated decay of the pH difference across vesicular membrane (delta pH) in the pH region 6-8 and salt (MCI, M+ = K+, Na+) concentration range 50-300 mM. 1) At low pH conditions (approximately 6), H+ transport through the gramicidin channel predominantly limits the delta pH decay rate. 2) At higher pH conditions (approximately 7.5), transport of a deprotonated species (but not through the channel) predominantly limits the rate. The second mechanism has been suggested to be the hydroxyl ion propogation through water chains across the bilayer by hydrogen bond exchange. In both mechanisms alkali metal ion transport providing the compensating flux takes place through the gramicidin channels. Such an identification has been made from a detailed study of the delta pH decay rate as a function of 1) gramicidin concentration, 2) alkali metal ion concentration, 3) pH, 4) temperature, and 5) changes in the membrane order (by adding small amounts of chloroform to vesicle solutions). The apparent activation energy associated with the second mechanism (approximately 3.2 kcal/mol) is smaller than that associated with the first mechanism (approximately 12 kcal/mol). In these experiments, delta pH was created by temperature jump, and vesicles were prepared using soybean phospholipid or a mixture of 94% egg phosphatidylcholine and 6% phosphatidic acid
Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H<sup>+</sup>/ M<sup>+</sup> exchange across phospholipid vesicular membranes
415-421From a study or the decay or the pH difference across vesicular membranes
(Δ pH) it has been possible to show that H+ and alkali metal ion (M+) concentration gradients across bilayer membranes (which are responsible for driving important biochemical processes) can be selectively perturbed by
anaesthetics such as chloroform and benzyl alcohol by combining them with a
suitable exchange ionophore. On adding the anaesthetic to the membrane in an environment
containing metal ions M+=K+. the rate or Δ pH
decay by H+/M+ exchange increases by a larger factor or by a smaller factor
(when compared to that in a membrane environment with M+=Na+) depending on whether the exchange
ionophore chosen is monensin or nigeriein. A rational explanation of this
"metal ion specificity" can be given using the exchange ionophore
mediated ion transport scheme in which the equilibrations at the
"interfaces" are fast compared to the "translocation equilibration"
between the species in the two layers of the membrane. The following three
factors are responsible for the observed "specificity": On adding the
anesthetic (i) translocation rate constants increase. (ii) the concentrations
of the M+ bound ionophores increase at the
expense of H+ bound ionophores. (iii) Under our
experimental conditions the rate determining species are the complexes
monensin-K (Mon-K) and nigeriein-H (Nig-H) for M+=K+ whereas they are monensin -H (MonH)
and nigeriein-Na (Nig-Na) for M+=Na+ Possible anesthetic induced membrane perturbations contributing
to the above mentioned changes in the membrane are (A), the loosening of the
membrane structure and (B ), an associated increase in the membrane hydration
(and membrane dielectric constant ). An analysis of the consequent changes in
the various transport steps shows the following: (a), The anaesthetic induced
changes in the translocation rates of electrically charged species are
not relevant in the explanation or
the observed changes in the Δ pH
decay rates. (b), Changes in the rates of fas<span style="font-size:
14.0pt;font-family:HiddenHorzOCR;mso-hansi-font-family:Arial;mso-bidi-font-family:
HiddenHorzOCR">t equilibria
at the interface contribute to changes in KH and <span style="font-size:14.0pt;font-family:HiddenHorzOCR;mso-hansi-font-family:Arial;
mso-bidi-font-family:HiddenHorzOCR">KM (c), A suggestion made in the
literature, that a significant interaction between the dipole moment of the
monensin-K complex and the membrane slows down its translocation, is not valid.
(d), The ability to explain rationally all the Δ p<span style="font-size:14.0pt;
font-family:HiddenHorzOCR;mso-hansi-font-family:Arial;mso-bidi-font-family:
HiddenHorzOCR">H decay
data confirms the validity or the transport scheme used. In our experiments Δ pH across the vesicular membrane was
created by pH jump coming from a
temperature jump.
</span
Binding site of the dye in bromophenol blue-lysozyme complex proton magnetic resonance study in aqueous solutions
The forward rate constant for the binding of Bromophenol blue to lysozyme is estimated to be 105M-1 s-1 at room temperature and ionic strength 0.01, from the line broadening of the 1H-NMR spectrum of Bromophenol blue at 270 MHz. The broadening of the 1H-NMR line corresponding to histidine-15 of lysozyme, in the presence of Bromophenol blue in solution at pH 4.5, suggests that the binding site of Bromophenol blue is not far from this protein residue. The extent of this broadening is pH-dependent and can be interpreted in terms of the average coulombic interaction between the dye and His-15. Since Bromophenol blue is known to inhibit lytic activity of lysozyme towards cell wall substrates, we can say that the region near His-15 may also be important for the efficient catalytic activity of lysozyme
Two mechanisms of nonactin mediated K<sup>+</sup> ion transport across phospholipid vesicular membranes
283-293From kinetic data it has been possible to
show that when a weak acid such as carbonyl cyanide m-chlorophenylhydrazone
(CCCP) is also present in phospholipid vesicles the ionophore nonactin (NON) transports
K+ ions across the membrane by two mechanisms: (I) As the charged species
NON-K+ and (II) as the electroneutral complex NON-K+- CCCP-.
In mechanism I, the anion CCCP- is also translocated across the membrane
as charged species such that the net charge translocated is zero. In the earlier
experiments using valinomycin (VAL) and CCCP (Prabhananda B S & Kombrabail M
H (1995) Biochim, Biophys, Acta 1235, 323-335) the existence
of a mechanism similar to mechanism I could not be detected. Even with NON (instead
of VAL), at the concentrations of our experiments mechanism II is dominant. The
relative dominance of mechanism II could be decreased either (i), by lowering the
ionophore concentration or (ii), by "catalysing" the transport of
CCCP- from the "polar region" to the "nonpolar region"
of the membrane by adding VAL at small concentrations. The kinetics of K+
transport used in arriving at such conclusions were inferred from the rate of decay
of H+ concentration difference (ΔpH) across phospholipid vesicular
membrane after creating ΔpH by temperature jump. A new procedure has been
used to estimate the translocation rate constant k+ of NON-K+
in soybean phospholipid vesicles (~1.5 × 103 s-1) by identifying
the NON-K+ translocation limited contribution to 1/τ by the combined
action of NON, CCCP and tetraphenylphosphonium ions (TPP+). The apparent
K+ dissociation constant of NON-K+ (~ 0.14 M), dissociation
constant of NON-K+- CCCP - in the membrane (~30mM),
and translocation rate constant of NON-K+- CCCP - (k0~
4.2×103 s-1) have been determined in soybean phospholipid
vesicle solutions containing 100 mM KCl
Evidence for dimer participation and evidence against channel mechanism in A23187-mediated monovalent metal ion transport across phospholipid vesicular membrane.
The decay of the pH difference (DeltapH) across soybean phospholipid vesicular membrane by ionophore A23187 (CAL)-mediated H+/M+ exchange (M+ = Li+, Na+, K+, and Cs+) has been studied in the pH range 6-7.6. The DeltapH in these experiments were created by temperature jump. The observed dependence of DeltapH relaxation rate 1/tau on the concentration of CAL, pH, and the choice of M+ in vesicle solutions lead to the following conclusions. 1) The concentrations of dimers and other oligomers of A23187 in the membrane are small compared to the total concentration of A23187 in the membrane, similar to that in chloroform solutions reported in the literature. 2) In the H+ transport cycle leading to DeltapH decay, the A23187-mediated H+ translocation across the membrane is a fast step, and the rate-limiting step is the A23187-mediated M+ translocation. 3) Even though the monomeric Cal-H is the dominant species translocating H+, Cal-M is not the dominant species translocating M+ (even at concentrations higher than [Cal-H]), presumably because its dissociation rate is much higher than its translocation rate. 4) The pH dependence of 1/tau shows that the dimeric species Cal2LiLi, Cal2NaNa, Cal2KH, and Cal2CsH are the dominant species translocating M+. The rate constant associated with their translocation has been estimated to be approximately 5 x 10(3) s-1. With this magnitude for the rate constants, the dimer dissociation constants of these species in the membrane have been estimated to be approximately 4, 1, 0.05, and 0.04 M, respectively. 5) Contrary to the claims made in the literature, the data obtained in the DeltapH decay studies do not favor the channel mechanism for the ion transport in this system. 6) However, they support the hypothesis that the dissociation of the divalent metal ion-A23187 complex is the rate limiting step of A23187-mediated divalent metal ion transport
Kinetics and mechanism of anionic ligand binding to carbonic anhydras
The kinetics of complex formation between Co(II)-carbonic anhydrase B and the anions cyanate, thiocyanate and cyanide has been studied at different pH values employing temperature-jump relaxation spectrometry. Formation of the 1 : 1 complex occurs via binding of the deprotonated state of the anion to an acidic state of the enzyme. The determined formation rate constants range from 108 to 3 × 109 M−1 s−1 and are two to three orders of magnitude higher than the value estimated for a ligand coordination to the central Co2+, based on a solvate substitution mechanism. These kinetic results strongly indicate that the deprotonated anion binds to an unoccupied coordination position of the protein-bound heavy metal ion in the form of an addition reaction. Upon binding of the anion, the coordination number of the Co2+ in the acidic state of the enzyme is increased from four to five. In the case of cyanide, a 2:1 anion complex is also formed. The formation rate constant is 5 × 105 M−1 s−1 which provides good evidence that this binding process is controlled by a solvate substitution mechanism