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PubMed1976

Light-scattering investigations of the subunit dissociation of human hemoglobin A. Effects of the aliphatic acid salts.

The subunit dissociation of human hemoglobin A by the aliphatic acid salts at neutral pH has been investigated by light-scattering molecular-weight measurements at 630 nm. Dissociation of hemoglobin tetramers to alphabeta dimers is observed in essentially all experiments at low to intermediate levels of salt concentrations, below the denaturation transitions, described in the accompanying paper (Ibanez, V.S., and Herskovits, T.T. (1976), Biochemistry 15, preceding paper in this issue). The effectiveness of the salts as subunit dissociating agent, reflected by the slopes, s, of the plots of deltaGDdegrees, the standard free energy of dissociation, vs. [D], the salt concentration, is found to increase with increasing alkyl chain length or hydrocarbon content of the salt. Estimates of the apparent number of amino acid sites at the areas of contact per alphabeta dimer formed, N', based on the slopes of the higher members of the series have been obtained using the equation, deltaGDdegrees = deltaGD,Wdegrees - 2N'RTKB[D]. Independent estimates of the binding constant, KB, required for these calculations were based on free-energy transfer data of hydrophobic amino acid alkyl groups and protein denaturation data. Our estimates of N' denaturation data. Our estimates of N' obtained with the more reliable data of the higher members of salt series are in the ranges of 19 and 27 amino acid groups, shown by the x-ray crystallographic structure of horse and human hemoglobin of Perutz (Perutz, M.F., et al. (1968), Nature (London) 219, 131) and Fermi ((1975) J. Mol. Biol. 97, 237) for the smaller alpha1 beta2 contact areas in the tetrameric structure. The lower estimates than 27 based on our dissociation of human hemoglobin suggest that several of the amino acid residues in the contact areas of the subunits are partially exposed to solvent. The increasing effectiveness of the higher mn imporant source of stabilization of the tetrameric structure of hemoglobin.

Binding Sites↗
PubMed2002

Dissociation of the GroEL-GroES asymmetric complex is accelerated by increased cooperativity in ATP binding to the GroEL ring distal to GroES.

A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance. Excellent fits of the data were obtained using a double-exponential equation with a linear drift. Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP. The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES. Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring. It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex.

Adenosine Triphosphate↗
PubMed2002

Reversible ligand-induced dissociation of a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus.

The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.5) for the substrate, fructose-6-phosphate (Fru-6-P), as compared to the wild-type enzyme. The dissociation constant for the inhibitor, phospho(enol)pyruvate (PEP), increases 2-fold, and the coupling parameter, Q(ay), decreases 2-fold. This suggests that while the mutant displays a slightly decreased affinity for PEP, PEP is still an effective inhibitor once bound. The new position of the tryptophan in W179Y/Y164W is approximately 6 A from the Fru-6-P portion of the active site. A 25% decrease in fluorescence intensity is observed upon Fru-6-P binding, and an 80% decrease in fluorescence intensity is observed with PEP binding. In addition, the intrinsic fluorescence polarization increases from 0.327 +/- 0.001 to 0.353 +/- 0.001 upon Fru-6-P binding, but decreases to 0.290 +/- 0.001 when PEP binds. Most notably, the presence of PEP induces dissociation of the tetramer. Dissociation of the tetramer into dimers occurs along the active site interface and can be monitored by the loss in activity or the loss in tryptophan fluorescence that is observed when the enzyme is titrated with PEP. Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.

Allosteric Site↗
PubMed1996

Deterministic pressure dissociation and unfolding of triose phosphate isomerase: persistent heterogeneity of a protein dimer.

Subunit dissociation and unfolding of dimeric rabbit muscle triose phosphate isomerase (TIM) induced by hydrostatic pressure were investigated. Changes in fluorescence emission of TIM (both intrinsic and of covalently attached probes) indicated that pressure ranging from 1 bar to 3.5 kbar promoted subunit dissociation and unfolding. Instrinsic fluorescence changes upon unfolding by pressure included a 27 nm red-shift of the emission, a decrease in fluorescence anisotropy from 0.14 to about 0.01, and a 1.5-fold increase in fluorescence quantum yield, similar to that observed in the presence of guanidine hydrochloride. Kinetics of pressure-induced fluorescence changes were slow (t 1/2 approximately 15 min) and little dependent on pressure. In order to selectively monitor subunit dissociation, fluorescence resonance energy transfer (FRET) measurements were carried out with TIM that was separately labeled with 5-((((2-iodoacetyl)-amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) or fluorescein-5-isothiocyanate (FITC). FRET measurements indicated that subunit dissociation and unfolding took place concomitantly, both under equilibrium conditions and in kinetic experiments in which dissociation/unfolding was triggered by a sudden increase in pressure. Release of pressure caused monomer refolding and dimerization. Contrary to what would be expected for a process involving subunit dissociation, pressure effects on TIM were not dependent on protein concentration. Experiments involving a series of pressure jumps demonstrated persistent heterogeneity in sensitivity toward pressure in the ensemble of TIM dimers. This kind of deterministic behavior is similar to that exhibited by higher order protein aggregates and indicates that not all individual dimers are energetically identical in solution. The heterogeneity of native TIM revealed by sensitivity to pressure could not be detected by traditional means of protein separation, such as polyacrylamide gel electrophoresis (under both native and denaturing conditions) and size exclusion gel chromatography. This suggests that energetic heterogeneity originates from conformational heterogeneity of the protein. The possible biological relevance of the deterministic character of stability of TIM is discussed.

Animals↗
PubMed1996

Dissociation kinetics of echinomycin from CpG binding sites in different sequence environments.

We have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 degrees C, echinomycin dissociates more slowly from CpG sites flanked by (AT)n (t1/2 approximately 40 min) and (CA)n.(TG)n (t1/2 approximately 11 min) than by An.Tn (t1/2 < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)4CG(TTA)4 also represents a very good binding site (t1/2 approximately 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)10(G/C)4(AT)10, the dissociation from CGGC is slower than that from CCCG or CCGC.

Antibiotics, Antineoplastic↗
PubMed1997

Evidence supporting a role for microfilaments in regulating the coupling between poorly dissociable IgE-Fc epsilonRI aggregates downstream signaling pathways.

Aggregation of Fc epsilonRI, the high-affinity receptor for IgE, on RBL-2H3 mast cells caused by reversible ligands such as multivalent antigen causes cellular responses that can be halted by subsequent addition of excess monovalent ligand. In contrast, Ca2+ and degranulation responses elicited by effectively irreversible streptavidin cross-linking of biotinylated IgE-Fc epsilonRI are not stopped by addition of excess biotin after stimulation is initiated. These results support previous conclusions based on studies with covalent oligomers of IgE that stable cross-links can continue to deliver stimulatory signals for extended periods of time. Dissociation measured in the presence of monovalent hapten reveals two populations of IgE-Fc epsilonRI cross-linked by multivalent antigen that differ in functional effectiveness. Aggregates with readily dissociable cross-links are normally responsible for triggering essentially all of the degranulation response, whereas aggregates with poorly dissociable cross-links apparently do not trigger this response. Treatment of RBL-2H3 cells with cytochalasin D, an inhibitor of actin polymerization, enhances downstream signaling and enables the less readily dissociable aggregates to stimulate Ca2+ and degranulation responses. Under these conditions, cytochalasin D does not affect hapten-mediated dissociation of multivalent antigen, nor does it prevent hapten from reversing tyrosine phosphorylation of Syk. Cytochalasin D alone causes tyrosine phosphorylation of a protein at approximately 75 kDa, and it reduces hapten-induced reversal of antigen-stimulated tyrosine phosphorylation of several other proteins. Taken together, these results indicate that stimulated actin polymerization normally regulates the coupling of aggregated Fc epsilonRI to downstream signaling pathways, and they provide an explanation for seeming discrepancies between responses to stable and reversible cross-links.

Actin Cytoskeleton↗
PubMed1999

A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition.

The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-peptide), leaving behind the membrane-bound A1.LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (Kd = 7 nM), suggesting that the membrane-bound LC.A1 product would act to sequester APC. This inhibitory interaction (LC.A1.APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.

Animals↗
PubMed1999

Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein.

Bacterial Proteins↗
PubMed2003

Kinetics and mechanism of the interaction of nitric oxide with pentacyanoferrate(II). Formation and dissociation of [Fe(CN)5NO]3 -.

The interaction of NO with [Fe(CN)(5)H(2)O](3)(-) (generated by aquation of the corresponding ammine complex) to produce [Fe(CN)(5)NO](3)(-) was studied by UV-vis spectrophotometry. The reaction product is the well characterized nitrosyl complex, described as a low-spin Fe(II) bound to the NO radical. The experiments were performed in the pH range 4-10, at different concentrations of NO, temperatures and pressures. The rate law was first-order in each of the reactants, with the specific complex-formation rate constant, k(f)( )()= 250 +/- 10 M(-)(1) s(-)(1) (25.4 degrees C, I = 0.1 M, pH 7.0), DeltaH(f)() = 70 +/- 1 kJ mol(-)(1), DeltaS(f)() = +34 +/- 4 J K(-)(1) mol(-)(1), and DeltaV(f)() = +17.4 +/- 0.3 cm(3) mol(-)(1). These values support a dissociative mechanism, with rate-controlling dissociation of coordinated water, and subsequent fast coordination of NO. The complex-formation process depends on pH, indicating that the initial product [Fe(CN)(5)NO](3)(-) is unstable, with a faster decomposition rate at lower pH. The decomposition process is associated with release of cyanide, further reaction of NO with [Fe(CN)(4)NO](2)(-), and formation of nitroprusside and other unknown products. The decomposition can be prevented by addition of free cyanide to the solutions, enabling a study of the dissociation process of NO from [Fe(CN)(5)NO](3)(-). Cyanide also acts as a scavenger for the [Fe(CN)(5)](3)(-) intermediate, giving [Fe(CN)(6)](4)(-) as a final product. From the first-order behavior, the dissociation rate constant was obtained as k(d) = (1.58 +/- 0.06) x 10(-)(5) s(-)(1) at 25.0 degrees C, I = 0.1 M, and pH 10.2. Activation parameters were found to be DeltaH(d)() = 106.4 +/- 0.8 kJ mol(-)(1), DeltaS(d)() = +20 +/- 2 J K(-)(1) mol(-)(1), and DeltaV(d)() = +7.1 +/- 0.2 cm(3) mol(-)(1), which are all in line with a dissociative mechanism. The low value of k(d) as compared to values for the release of other ligands L from [Fe(II)(CN)(5)L](n)()(-) suggests a moderate to strong sigma-pi interaction of NO with the iron(II) center. It is concluded that the release of NO from nitroprusside in biological media does not originate from [Fe(CN)(5)NO](3)(-) produced on reduction of nitroprusside but probably proceeds through the release of cyanide and further reactions of the [Fe(CN)(4)NO](2)(-) ion.

Journal Article↗
PubMed2003

Bond dissociation energy and Lewis acidity of the xenon fluoride cation.

This study focuses upon the Lewis acid reactivity of XeF(+) with various bases in the gas phase and the determination of the bond dissociation energy of XeF(+). The bond dissociation energy of XeF(+) has been measured by using energy-resolved collision-induced dissociation with neon, argon, and xenon target gases. Experiments with neon target yield a 298 K bond dissociation enthalpy of 2.81 +/- 0.09 eV, and those with argon target give a similar value at 2.83 +/- 0.12 eV. When using a xenon target, a significantly lower value of 1.95 +/- 0.16 eV was observed, which corresponds closely with previous measurements and theoretical predictions. It is proposed that the lighter target gases give inefficient excitation of the XeF(+) vibration leading to dissociation at energies higher than the BDE. Novel xenon-base adducts have been prepared in a flowing afterglow mass spectrometer by termolecular addition to XeF(+) and by reaction of base with XeF(+)(H(2)O). New species have been characterized qualitatively by CID, and it is found that the products formed reflect the relative ionization energies of the fragments. Among the new xenon-containing species that have been prepared are the first examples of xenon carbonyls.

Journal Article↗
PubMed2004

Toward a photochemical and thermal molecular machine: reversible ligand dissociation and binding in a ruthenium(II)-2,2'-bipyridine complex with tris(2-pyridylmethyl)amine.

A Ru(II) complex having tris(2-pyridylmethl)amine (TPA) and 2,2'-bipyridine (bpy), [Ru(TPA)(bpy)]X(2) (X = ClO(4), PF(6)), exhibited a severe distortion of the coordination of the axial pyridine moiety of TPA due to steric hindrance. The complex showed interesting dissociation-binding behavior of the axial pyridine arm to form a solvent adduct with TPA ligation in a unique meridional tridentate fashion. The complex undergoes thermal dissociation to form solvent-coordinated species via an S(N)2-like mechanism with activation energy of 117 kJ/mol. In contrast, the complex showed reversible photochemical dissociation and rebinding via an S(N)1-like mechanism by MLCT irradiation. The photochemical dissociation was accelerated approximately 200-fold faster than the thermal process. The dissociation process involves selective binding behavior toward external ligands (solvents) with pi-acceptor character, which is indispensable, and no sigma-donating molecules could bind to the Ru(II) center. The guest molecule can be released upon photoirradiation after its thermal binding.

Journal Article↗
PubMed2001

Trapping-mediated dissociative chemisorption of cycloalkanes on Ru(001) and Ir(111): influence of ring strain and molecular geometry on the activation of C-C and C-H bonds.

We have measured the initial probabilities of dissociative chemisorption of perhydrido and perdeutero cycloalkane isotopomers on the hexagonally close-packed Ru(001) and Ir(111) single-crystalline surfaces for surface temperatures between 250 and 1100 K. Kinetic parameters (activation barrier and preexponential factor) describing the initial, rate-limiting C-H or C-C bond cleavage reactions were quantified for each cycloalkane isotopomer on each surface. Determination of the dominant initial reaction mechanism as either initial C-C or C-H bond cleavage was judged by the presence or absence of a kinetic isotope effect between the activation barriers for each cycloalkane isotopomer pair, and also by comparison with other relevant alkane activation barriers. On the Ir(111) surface, the dissociative chemisorption of cyclobutane, cyclopentane, and cyclohexane occurs via two different reaction pathways: initial C-C bond cleavage dominates on Ir(111) at high temperature (T > approximately 600 K), while at low temperature (T < approximately 400 K), initial C-H bond cleavage dominates. On the Ru(001) surface, dissociative chemisorption of cyclopentane occurs via initial C-C bond cleavage over the entire temperature range studied, whereas dissociative chemisorption of both cyclohexane and cyclooctane occurs via initial C-H bond cleavage. Comparison of the cycloalkane C-C bond activation barriers measured here with those reported previously in the literature qualitatively suggests that the difference in ring-strain energies between the initial state and the transition state for ring-opening C-C bond cleavage effectively lowers or raises the activation barrier for dissociative chemisorption via C-C bond cleavage, depending on whether the transition state is less or more strained than the initial state. Moreover, steric arguments and metal-carbon bond strength arguments have been evoked to explain the observed trend of decreasing C-H bond activation barrier with decreasing cycloalkane ring size.

Journal Article↗
PubMed2001

An insight into alkali promotion: a density functional theory study of CO dissociation on K/Rh(111).

The important role of alkali additives in heterogeneous catalysis is, to a large extent, related to the high promotion effect they have on many fundamental reactions. The wide application of alkali additives in industry does not, however, reflect a thorough understanding of the mechanism of their promotional abilities. To investigate the physical origin of the alkali promotion effect, we have studied CO dissociation on clean Rh(111) and K-covered Rh(111) surfaces using density functional theory. By varying the position of potassium atoms relative to a dissociating CO, we have mapped out the importance of different K effects on the CO dissociation reactions. The K-induced changes in the reaction pathways and reaction barriers have been determined; in particular, a large reduction of the CO dissociation barrier has been identified. A thorough analysis of this promotion effect allows us to rationalize both the electronic and the geometrical factors that govern alkali promotion effect: (i) The extent of barrier reductions depends strongly on how close K is to the dissociating CO. (ii) Direct K-O bonding that is in a very short range plays a crucial role in reducing the barrier. (iii) K can have a rather long-range effect on the TS structure, which could reduce slightly the barriers.

Journal Article↗
PubMed2003

Origin of asymmetric charge partitioning in the dissociation of gas-phase protein homodimers.

The origin of asymmetric charge and mass partitioning observed for gas-phase dissociation of multiply charged macromolecular complexes has been hotly debated. These experiments hold the potential to provide detailed information about the interactions between the macromolecules within the complex. Here, this unusual phenomenon of asymmetric charge partitioning is investigated for several protein homodimers. Asymmetric charge partitioning in these ions depends on a number of factors, including the internal energy, charge state, and gas-phase conformation of the complex, as well as the conformational flexibility of the protein monomer in the complex. High charge states of both cytochrome c and disulfide-reduced alpha-lactalbumin homodimers dissociate by a symmetrical charge partitioning process in which both fragment monomers carry away roughly an equal number of charges. In contrast, highly asymmetric charge partitioning dominates for the lower charge states. Cytochrome c dimer ions with eleven charges formed by electrospray ionization from two solutions in which the solution-phase conformation differs dissociate with dramatically different charge partitioning. These results demonstrate that these gas-phase complexes retain a clear "memory" of the solution from which they are formed, and that information about their solution-phase conformation can be obtained from these gas-phase dissociation experiments. Cytochrome c dimer ions formed from solutions in which the conformation of the protein is native show greater asymmetric charge partitioning with increasing ion internal energy. Cytochrome c dimers that are conformationally constrained with intramolecular cross-linkers undergo predominantly symmetric charge partitioning under conditions where asymmetric charge partitioning is observed for cytochrome c dimers without cross-links. Similar results are observed for alpha-lactalbumin homodimers. These results provide convincing evidence that the origin of asymmetric charge partitioning in these homodimers is the result of one of the protein monomers unfolding in the dissociation transition state. A mechanism that accounts for these observations is proposed.

Cytochrome c Group↗
PubMed2006

Force-induced insulin dimer dissociation: a molecular dynamics study.

Understanding the forces and dynamics of insulin dissociation is critical for devising formulations for the treatment of insulin-dependent diabetes. In earlier work, we applied AFM-based force spectroscopy to covalently tethered and oriented insulin monomers to assess the effect of molecular orientation on insulin-insulin binding forces. We report here on steered molecular dynamics simulations of the insulin dissociation force spectroscopy experiment. Consistent with our experiments, our simulation results suggest that insulin dimer dissociation occurs near the limit of extensibility of the B-chain. We have also found that the forced dissociation of the insulin dimer is a rate-dependent process, involving significant conformational changes to the monomer(s). The insulin dimer dissociation pathway also depends on the relative strength of the inter-monomer interactions across the antiparallel beta-sheet interface and the intra-monomer interactions of residues A1 and A30 with the insulin B-chain. Our simulation results strongly support the design of bioactive insulin analogues that involves altering hydrogen bonding and hydrophobic interactions across the beta-sheet dimer interface.

Dimerization↗
PubMed2001

Mechanism for the ethanol-dependent heat-induced dissociation of casein micelles.

An explanation as to how casein micelles dissociate when heated in the presence of ethanol is presented. Dissociation of casein micelles in milk-ethanol mixtures was studied using (1)H NMR, and the effects of addition of CaCl(2), NaCl, or EDTA or alteration of milk pH on this dissociation were studied. It is proposed that at low temperatures, ethanol reduces the solvent quality of milk serum, but above a critical temperature (approximately 30 degrees C in a 35% ethanol solution), ethanol enhances solvent quality and dissociates the casein micelles. Ethanol reduced protein hydrophobicity and increased the pK(a) value of phosphoserine, effects that are likely to be significant in the dissociating effect of ethanol at elevated temperatures.

Animals↗
PubMed2006

Homolytic dissociation of the vulcanization accelerator tetramethylthiuram disulfide (TMTD) and structures and stabilities of the related radicals Me2NCSn* (n = 1-4).

The homolytic dissociation of the important vulcanization accelerator tetramethylthiuram disulfide (TMTD) has been studied by ab initio calculations according to the G3X(MP2) and G3X(MP2)-RAD theories. Homolytic cleavage of the SS bond requires a low enthalpy of 150.0 kJ mol-1, whereas 268.0 kJ mol-1 is needed for the dissociation of one of the C-S single bonds. To cleave one of the SS bonds of the corresponding trisulfide (TMTT) requires 191.1 kJ mol-1. Me2NCS2* is a particularly stable sulfur radical as reflected in the low S-H bond dissociation enthalpy of the corresponding acid Me2NC(=S)SH (301.7 kJ mol-1). Me2NCS2* (2B2) is a sigma radical characterized by the unpaired spin density shared equally between the two sulfur atoms and by a 4-center (NCS2) delocalized pi system. The ESR g-tensors of the radicals Me2NCSn* (n = 1-3) have been calculated. Both TMTD and the mentioned radicals form stable chelate complexes with a Li+ cation, which here serves as a model for the zinc ions used in accelerated rubber vulcanization. Although the binding energy of the complex [Li(TMTD)]+ is larger than that of the isomeric species [Li(S2CNMe2)2]+ (12), the dissociation enthalpy of TMTD as a ligand is smaller (125.5 kJ mol-1) than that of free TMTD. In other words, the homolytic dissociation of the SS bonds of TMTD is facilitated by the presence of Li+ ions. The sulfurization of TMTD in the presence of Li+ to give the paramagnetic complex [Li(S3CNMe2)2]+ is strongly exothermic. These results suggest that TMTD reacts with naked zinc ions as well as with the surface atoms of solid zinc oxide particles in an analogous manner producing highly reactive complexes, which probably initiate the crosslinking process during vulcanization reactions of natural or synthetic rubber accelerated by TMTD/ZnO.

Journal Article↗
PubMed1996

Reactions of Charged Substrates. 4. The Gas-Phase Dissociation of (4-Substituted benzyl)dimethylsulfoniums and -pyridiniums.

The relative rates for the gas-phase dissociation RX(+) --> R(+) + X degrees of five (4-Y-substituted benzyl)dimethysulfoniums (Y = MeO, Me, H, Cl, and NO(2)) and 24 (4-Y-substituted benzyl)-3'-Z-pyridiniums (complete series for Z = CN, Cl, CONH(2), and H, and 4-methoxy- and 4-nitrobenzyls for Z = F and CH(3)CO) were measured using liquid secondary ion mass spectrometry. The Hammett plot (vs deltaDeltaG degrees or sigma(+)) is linear for the sulfoniums, but plots for the four pyridinium series have a drastic break between the 4-Cl and 4-NO(2) substrates. Brønsted-like plots for the pyridiniums show a strong leaving group effect only for 4-nitrobenzyls. An analysis of these linear free energy relations with supporting evidence from semiempirical computations suggests that collisionally activated pyridinium substrates dissociate by two pathways, direct dissociation and through an ion-neutral complex intermediate. Comparison of these results with results for the solution reactions of some of these compounds shows that the mechanism is different in the gas and solution phases. Sufficient experimental data are not available to assign a mechanism for dissociation to the sulfonium series, but computational results show characteristics of a direct dissociative mechanism.

Journal Article↗