Differences In Dissociation Rates Research Articles

FimH-mannose noncovalent bonds survive minutes to hours under force

The adhesin FimH is expressed by commensal Escherichia coli and is implicated in urinary tract infections, where it mediates adhesion to mannosylated glycoproteins on urinary and intestinal epithelial cells in the presence of a high-shear fluid environment. The FimH-mannose bond exhibits catch behavior in which bond lifetime increases with force, because tensile force induces a transition in FimH from a compact native to an elongated activated conformation with a higher affinity to mannose. However, the lifetime of the activated state of FimH has not been measured under force. Here we apply multiplexed magnetic tweezers to apply a preload force to activate FimH bonds with yeast mannan, then we measure the lifetime of these activated bonds under a wide range of forces above and below the preload force. A higher fraction of FimH-mannan bonds were activated above than below a critical preload force, confirming the FimH catch-bond behavior. Once activated, FimH detached from mannose with multi-state kinetics, suggesting the existence of two bound states with a 20-fold difference in dissociation rates. The average lifetime of activated FimH-mannose bonds was 1000 to 10,000 s at forces of 30–70 pN. Structural explanations of the two bound states and the high force resistance provide insights into structural mechanisms for long-lived, force-resistant biomolecular interactions.

Identification of Individual Immobilized DNA Molecules by Their Hybridization Kinetics Using Single-Molecule Fluorescence Imaging.

Single-molecule fluorescence methods can count molecules without calibration, measure kinetics at equilibrium, and observe rare events that cannot be detected in an ensemble measurement. We employ total internal reflection fluorescence microscopy to monitor hybridization kinetics between individual spatially resolved target DNA molecules immobilized at a glass interface and fluorescently labeled complementary probe DNA in free solution. Using super-resolution imaging, immobilized target DNA molecules are located with 36 nm precision, and their individual duplex formation and dissociation kinetics with labeled DNA probe strands are measured at site densities much greater than the diffraction limit. The purpose of this study is to evaluate uncertainties in identifying these individual target molecules based on their duplex dissociation kinetics, which can be used to distinguish target molecule sequences randomly immobilized in mixed-target samples. Hybridization kinetics of individual target molecules are determined from maximum likelihood estimation of their dissociation times determined from a sample of hybridization events at each target molecule. The dissociation time distributions thus estimated are sufficiently narrow to allow kinetic discrimination of different target sequences. For example, a single-base thymine-to-guanine substitution on immobilized strands produces a 2.5-fold difference in dissociation rates of complementary probes, allowing for the identification of individual target DNA molecules by their dissociation rates with 95% accuracy. This methodology represents a step toward high-density single-molecule DNA microarray sensors and a powerful tool to investigate the kinetics of hybridization at surfaces at the molecular level, providing information that cannot be acquired in ensemble measurements.

Molecular dynamics study on the dissociation of methane hydrate via inorganic salts

ABSTRACTHydrate plugging is a hidden threat to the safe exploitation of oil and gas. Inorganic salts are widely used as thermodynamic inhibitors to effectively prevent the hydrate formation. This study uses a molecular dynamics method to explore the mechanism of the hydrate dissociation via inorganic salts on the micro-scale. We simulated the dissociating process of methane hydrate under different concentration series of NaCl, KCl and CaCl2 solutions at 273 K, and analysed the changes of ionic structure, transport parameters and kinetic energy in the system of inorganic salt/hydrate. The simulation results successfully revealed the step-by-step dissociation of hydrate, and the differences in dissociation rates among the different inhibitors. The energy needed for hydrate dissociation alters for different inorganic solutions; the energy reaches maximum when KCl is the inhibitor, and lowest when the concentration of CaCl2 exceeds 30% w/w. We calculated the coordination numbers of all components, including oxygen atoms, cations and anions, and also their diffusion coefficients; analysed the effects of the three inorganic salts on the simulated hydrate structure and its transport; in addition, investigated the mechanism of hydrate dissociation via inorganic salts.

Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding.

The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight.

A sequence-specific threading tetra-intercalator with an extremely slow dissociation rate constant

A long-lived and sequence specific ligand-DNA complex would make possible the modulation of biological processes for extended periods. We have been investigating the threading polyintercalation approach to DNA recognition in which chains of aromatic units thread back and forth repeatedly through the double helix. Here we report the preliminary sequence specificity and detailed kinetic analysis of a structurally characterized threading tetraintercalator. Specific binding on a relatively long DNA strand was observed, strongly favoring a predicted 14-base pair sequence. Kinetic studies revealed a multi-step association process and specificity was found to derive primarily from large differences in dissociation rates. Importantly, the rate-limiting dissociation rate constant of the tetraintercalator complex dissociating from its preferred binding site was extremely slow, corresponding to a 16 day half-life, making it one of the longer non-covalent complex half-lives yet measured, and, to the best of our knowledge, the longest for a DNA binding molecule.

Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism—Importance of slow dissociation and relaxation rates for antiviral efficacy

The importance of slow dissociation of non-nucleoside reverse transcriptase inhibitors (NNRTIs) for antiviral effect has been investigated. The kinetic characteristics of a series of NNRTIs interacting with wild type and drug resistant variants of HIV-1 RT (EC 2.7.7.49) were analyzed by SPR biosensor technology. The antiviral effect was determined in MT-4 and peripheral blood mononuclear cells. Due to extremely slow dissociation rates and a complex interaction mechanism, rate constants could not be quantified. Instead, interaction characteristics were qualitatively analyzed using simulated sensorgrams. The simplest model describing these interactions adequately was an induced fit mechanism, i.e. a mechanism involving the formation of an initial enzyme-inhibitor complex subsequently transformed into a more stable complex. Differences in rates of dissociation from the initial complex and rates of relaxation from the induced complex explained (1) the differences in the amounts of formed complex, (2) the stability of the complex and (3) the antiviral efficacies of the compounds. The effect of NNRTI binding site mutations also correlated with these kinetic characteristics. MIV-170 was the most effective inhibitor of wild type and mutant HIV-1 in cell culture, a property that was associated with the formation of the largest amount of complex and the slowest relaxation and dissociation rates. This study supports the hypothesis that the efficacy of anti-HIV drugs is dependent on slow dissociation from the target, thereby maximizing the duration of the inhibitory effect. It also illustrates the strength of simulating interaction data for qualitative analysis of tight-binding drugs and the importance of resolving the kinetic mechanism of drug–target interactions.

Leukocyte Function-associated Antigen 1-mediated Adhesion Stability Is Dynamically Regulated through Affinity and Valency during Bond Formation with Intercellular Adhesion Molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.

Fidelity in protein synthesis

Fidelity in protein synthesis

Enantioselective detection of chiral phosphorescent analytes in cyclodextrin complexes

Inclusion complexes between camphorquinone (CQ) and cyclodextrins (CDs) in deoxygenated aqueous solutions are shown to exhibit relatively strong room temperature phosphorescence (RTP). Among the various CDs tested, α-CD showed the strongest RTP signals. Interestingly, these signals differed significantly for the two enantiomers of CQ; the phosphorescence lifetime of (+)-CQ was about four times longer than that of (−)-CQ, being 352 ± 16 and 89 ± 6 μs, respectively. This enantiomeric selectivity is attributed to a difference in dissociation rates (competing with the radiative emission process) for the diastereoisomeric inclusion complexes dealt with, which have a 2:1 stoichiometry (α-CD:CQ:α-CD). Time-resolved RTP detection using different delay times enables the determination of the two enantiomers in a mixture without involving a separation technique. The minimum detectable fraction of (+)-CQ in a 2 mM sample was 13%.

Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.

Kinetic determinants of agonist action at the recombinant human glycine receptor.

The amino acids glycine, beta-alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady-state single-channel recordings was used to compare the actions of glycine, beta-alanine and taurine upon homomeric alpha1 human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10-90 % rise times determined from rapid application of 100 microM of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 mM) the rise time for glycine (0.26 ms) was 1.8-fold faster than that for beta-alanine (0.47 ms) and 3.9-fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for beta-alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > beta-alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single-opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor.

Subtle Differences in Dissociation Rates of Interactions between Destabilized Human Carbonic Anhydrase II Mutants and Immobilized Benzenesulfonamide Inhibitors Probed by a Surface Plasmon Resonance Biosensor

The development of commercial biosensors based on surface plasmon resonance has made possible careful characterization of biomolecular interactions. Here, a set of destabilized human carbonic anhydrase II (HCA II) mutants was investigated with respect to their interaction kinetics with two different immobilized benzenesulfonamide inhibitors. Point mutations were located distantly from the active site, and the destabilization energies were up to 23 kJ/mol. The dissociation rate of wild-type HCA II, as determined from the binding to the inhibitor with higher affinity, was 0.019 s−1. For the mutants, dissociation rates were faster (0.022–0.025 s−1), and a correlation between faster dissociation and a high degree of destabilization was observed. We interpreted these results in terms of increased dynamics of the tertiary structures of the mutants. This interpretation was supported by entropy determinations, showing that the entropy of the native structure significantly increased upon destabilization of the protein molecule. Our findings demonstrate the applicability of modern biosensor technology in the study of subtle details in molecular interaction mechanisms, such as the long-range effect of point mutations on interaction kinetics.

Kinetic Analysis of the Interaction between HIV-1 Protease and Inhibitors Using Optical Biosensor Technology

The interaction between HIV-1 protease and reversible inhibitors was studied by surface plasmon resonance biosensor technology. The steady-state binding level and the time course of association and dissociation could be observed by measuring the binding of inhibitors injected in a continuous flow of buffer to the immobilized enzyme. Fourteen low molecular weight inhibitors (500–700 Da), including the four clinically used HIV-1 protease inhibitors (indinavir, nelfinavir, ritonavir, and saquinavir), were analyzed. Affinities were estimated as B50 values from a series of sensorgrams at different concentrations of inhibitors. These values were found to be correlated with inhibition constants (Ki) determined by an enzyme inhibition assay (r2 = 0.84, logarithmic values). Dissociation rates were estimated at a single saturating concentration of the inhibitors as t1/2,obs, but these values did not correlate with Ki (r2 = 0.26, logarithmic values). Indinavir had the highest affinity (B50 = 11 nM) and the fastest dissociation (t1/2,obs = 500 s) among the clinically used inhibitors while saquinavir had a lower affinity (B50 = 25 nM) and the slowest dissociation rate (t1/2,obs = 6500 s). Since these two inhibitors have similar Ki values, the differences in dissociation rates reveal important characteristics in the interaction that cannot be obtained by the inhibition studies. The biosensor data are expected to be of greater in vivo relevance since the experiments were performed in a buffer more similar to physiological conditions.

State-to-state unimolecular reaction dynamics of HOCl near the dissociation threshold: The role of vibrations, rotations, and IVR probed by time- and eigenstate-resolved spectroscopy

We use infrared-visible double resonance overtone excitation to prepare HOCl molecules in single, well-characterized rotational levels of high OH stretching states just above the dissociation threshold on the ground potential energy surface. Combined with time-resolved laser induced fluorescence (LIF) detection of the OH product, this approach allows us to monitor the dependence of unimolecular dissociation rate on the angular momentum (J,Ka,Kc), total energy, and vibrational character of the state of the reactant molecule as well as on number of dissociation channels available to the OH product. Dissociation rates from single states of the parent molecule are distributed over more than two orders-of-magnitude in a fashion that appears largely independent of the excess energy and the total angular momentum. In several instances we observe a one-order-of-magnitude difference in dissociation rate between states that are nearby in rotational quantum number and/or energy. Superimposed on these state-to-state rate fluctuations is a general trend toward decreasing unimolecular dissociation rate with increasing Ka quantum number. Moreover, the measured rates, which range from 1 to 300 μs−1, are much slower than the predictions of statistical theories. We present a simple model calculation to explain the observed phenomena.

Repertoire shift occurs during the memory maintenance phase of the immune responses and is not affinity-driven.

We have examined the immunoglobulin variable gene usage and antibody affinities during the memory maintenance phase of the immune response to the hapten phenyl-oxazolone. Hapten-specific hybridomas representing the memory population were generated 4-6 months postimmunization. The V-gene expression of these hybridomas was determined by reverse transcriptase-polymerase chain reaction screening and antibody affinities were estimated by biointeraction analysis (BIA) using the BIAcore biosensor. Our results show that the V-gene repertoire has already been shifted during the memory maintenance phase of the immune response, i.e. prior to a second antigenic challenge, and did not entail any advantages in terms of antigen-binding capacities. Our results concur with the view that antibody affinities are modulated mainly through differences in dissociation rates rather than in association rates, and the implications of this with respect to affinity maturation is discussed.

Physical parameters and biological stability of yttrium(III) diethylenetriaminepentaacetic acid derivative conjugates.

The solution equilibria, acid dissociation, and serum stability of a series of Y(III) complexes of DTPA ligands functionalized with p-nitrobenzyl, methyl, and trans-cyclohexyl substituents were studied. The thermodynamic stability of the complexes studied ranged from log K = 21.53 to 24.7. Acid dissociation rates were found to decrease as the substitution on the carbon backbone increased, and significant differences in dissociation rates were observed for the Y(III) complexes of a pair of diasteriomeric cyclohexyl-DTPA ligands. While one diastereomer was found to have the slowest acid dissociation rate of the entire DTPA series, it was remarkably labile in both serum stability and in vivo studies.

Moderate discrimination of REP-1 between Rab7⋅GDP and Rab7⋅GTP arises from a difference of an order of magnitude in dissociation rates

The kinetics of the interaction of Rab7 with REP-1 have been investigated using the fluorescence of GDP and GTP analogs at the active site of Rab7. The results show that REP-1 has higher affinity for the GDP bound form of Rab7 ( K d=1 nM) than for the GTP bound form ( K d=20 nM). Both affinities should still be sufficient for the formation of stable complexes in the cell. The association reaction proceeds in two steps for the GDP bound form. The initial step is fast ( k +1=ca. 10 7 M −1 s −1) and concentration dependent while the second represents a slow equilibration ( k +2+ k −2=3.5 s −1) which has little effect on the overall equilibrium. The difference in affinity of the two nucleotide bound forms arises from a difference in dissociation rates (0.012 s −1 for Rab7⋅GDP and 0.2 s −1 for Rab7⋅GTP).

Kinetics of nitric oxide dissociation from five- and six-coordinate nitrosyl hemes and heme proteins, including soluble guanylate cyclase.

Kinetics of NO dissociation were characterized for three five-coordinate systems, heme-NO, HSA-heme-NO (human serum albumin), GC-NO (soluble guanylate cyclase), and for the six-coordinate system, Im-heme-NO. Nitrosyl myoglobin was redetermined for comparison. Previously known, six-coordinate R and T state nitrosyl hemoglobins are also included in the comparison. The data indicate that NO dissociates more than 1000 times faster from five-coordinate model heme than it does from the six-coordinate analog. Such a negative trans-effect between NO and a proximal base is in sharp contrast to carboxy heme derivatives, in which ligand dissociation rates are greatly slowed in when a trans base is present. As a result of opposite trans-effects, six-coordinate carboxy and nitrosyl derivatives have comparable dissociation rates, even though the five-coordinate species are very different. In proteins, five- and six-coordinate forms do not show a large difference in dissociation rates. Part of the reason may be due to different probabilities for geminate recombination in the different proteins, but this cannot explain all the facts. There must also be influences of the protein structure on bond-breaking rate constants themselves. With the exception of hemoglobin in the T state, nitrosyl guanylate cyclase shows the highest NO dissociation rate constant, k(obs) = 6 x 10(-4) s(-1). This would yield a half-life of about 2 min at 37 degrees C for dissociation of NO from GC-NO, a number that has implications for the mechanism of regulation of the activity of this key heme enzyme.

Unsubstituted CyclidenesA Novel Family of Lacunar Dioxygen Carriers with Enhanced Stability toward Autoxidation: Synthesis, Characterization, and a Representative X-ray Structure

A major advance has been made in the incremental molecular design of long-lived cobalt(II) dioxygen carriers. Preceding mechanistic studies revealed that ionizable methyl groups trigger the autoxidation of the O2 adduct of the cobalt(II) cyclidene. Two members of a new family of unsubstituted (no methyl groups) lacunar cyclidene dioxygen carriers have been prepared in an eight-step synthesis, and a complex with a hexamethylene bridge has been structurally characterized. In contrast to previously studied cyclidenes, these materials bear no substituents on the chelated macrocyclic platform. As anticipated, the rates of autoxidation of these unsubstituted cyclidene complexes were found to be 5−8 times slower than those for the most stable previously known cyclidene derivatives. Because of the absence of Me−Me vicinal repulsion, the C6 bridge assumes a zig-zag conformation directly across the cavity. The accompanying, relatively low, dioxygen affinity is explained on the basis of electronic and steric factors. The rates of dioxygen binding to these newly prepared cobalt(II) unsubstituted cyclidenes are fast and approximately equal to the corresponding values for their Me-substituted analogs. Consequently, differences in dissociation rates are responsible for the differences in O2 affinities. This is a clear example of an unusual steric effect for O2 adducts.

Equilibria, kinetics and mechanism of complexation of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin and its N-methylated derivative with cadmium(II) and zinc(II) ions in aqueous solution at various temperatures and pressures. Effects of metal ion size and porphyrin ring deformation on metal ion incorporation

The equilibria and kinetics of Cd(II) and Zn(II) incorporation into water-soluble tetrakis(4-sulfonatophenyl)porphyrin (TPPS 4) and its N-methylated derivative ( N-MeTPPS 4) in aqueous solution were investigated at I=0.10 M (NaNO 3) at various temperatures and pressures. Thermodynamic and kinetic parameters for formation and dissociation of the metalloporphyrins were obtained. The equilibrium constants of the complexation of TPPS 4 with Cd(II) and Zn(II) ions are K Cd P=1.15×10 −10 M (Δ H°=37.3 kJ mol −1, Δ S°=−66 J mol −1 K −1) and K Zn P=3.7×10 −1 M (Δ H°=23.4 kJ mol −1, Δ S°=60 J mol −1 K −1), respectively, at 25.0°C. The equilibrium constant for Zn(II) ion is by ∼9 orders of magnitude larger than that for Cd(II) ion. In the case of N-MeTPPS 4 the equilibrium constants at 25.0°C are K Cd N -MeP=(1.16±0.03)×10 −2 (Δ H°=26.2±2.0 kJ mol −1, Δ S°=51±6 J mol −1 K −1) and K Zn N-MeP =(2.87±0.18)×10 (Δ H°=34.6±0.7 kJ mol −1, Δ S°=144±3 J mol −1 K −1), respectively. The difference in the equilibrium constant between Cd(II) and Zn(II) ions reduces by ∼10 3 from TPPS 4 to N-MeTPPS 4 due to the steric influence of the N-methyl group. The rate constants at 25.0°C are as follows: for the reaction between Cd(II) ion and TPPS 4 k f=5.21×10 2 M −1 s −1 (Δ H f ≠=49.5±3.1 kJ mol −1, Δ S f ≠=−27±2 J mol −1 K −1, Δ V f ≠=6.4±0.3 cm 3 mol −1) and k d=5.49×10 12 M −2 s −1 (Δ H d ≠=12.2±1.5 kJ mol −1, Δ S d ≠=39±5 J mol −1 K −1); for the reaction between Zn(II) ion and TPPS 4 k f=1.13 M −1 s −1 (Δ H f ≠=57.8±2.4 kJ mol −1, Δ S f ≠=−50±3 J mol −1 K −1, Δ V f ≠=3.9±0.3 cm 3 mol −1) and k d=9.37 M −2 s −1 (Δ H d ≠=34.4±1.5 kJ mol −1, Δ S d ≠=−110±3 J mol −1 K −1); for the reaction between Cd(II) and N-MeTPPS 4 k f=2.53×10 5 M −1 s −1 (Δ H f ≠=37.9±1.0 kJ mol −1, Δ S f ≠=−15±3 J mol −1 K −1, Δ V f ≠=−1.0±0.4 cm 3 mol −1) and k d=1.96×10 7 M −1 s −1 (Δ H d ≠=14.3±0.8 kJ mol −1, Δ S d ≠=−57±3 J mol −1 K −1, Δ V d ≠=−9.6±0.5 cm 3 mol −1); for the reaction between Zn(II) ion and N-MeTPPS 4 k f=2.10×10 3 M −1 s −1 (Δ H f ≠=54.6±0.9 kJ mol −1, Δ S f ≠=2±3 J mol −1 K −1, Δ V f ≠=2.8±0.9 cm 3 mol −1) and k d=6.37×10 M −1 s −1 (Δ H d ≠=19.3±1.5 kJ mol −1, Δ S d ≠=−146±5 J mol −1 K −1). Enhancement in rate for the metal ion incorporation by the methylation of the porphyrin is almost the same for both metal ions, i.e. by a factor of ∼10 3. The difference in dissociation rate between the Zn(II) and Cd(II) porphyrins is smaller for N-MeTPPS 4 than for TPPS 4, which results from the larger difference in the stability constant of the TPPS 4 complexes relative to the N-MeTPPS 4 complexes.