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PubMed1997

Theoretical and experimental considerations of the pseudo-first-order approximation in conventional kinetic analysis of IAsys biosensor data.

The validity of the conventional interpretation of IAsys biosensor profiles in terms of pseudo-first-order kinetic behavior is subjected to closer scrutiny by its application to simulated data for low- and high-affinity interactions between ligate and immobilized ligand. As might reasonably have been expected, analysis of the simulated data for the low-affinity system (association equilibrium constant of 10(5) M-1) in such terms returned the input association and dissociation rate constants (10(3) M-1 s-1 and 10(-2) s-1, respectively)-a consequence of essential compliance with the assumed constancy of ligate concentration in the liquid phase. For the high-affinity interaction (ka = 10(5) M-1 s-1, kd = 10(-2) s-1, KAX = 10(7) M-1) the ligate concentration was depleted by up to 35%, and hence its assumed constancy was clearly an untenable approximation. Whereas no symptomatic evidence of such violation (apart from the return of incorrect estimates of ka and kd) was evident from pseudo-first-order kinetic analysis of the adsorption profiles, the corresponding analysis of desorption profiles was more informative in that the data deviated demonstrably from pseudo-first-order kinetic behavior. A second-order kinetic analysis was therefore developed and shown to be applicable to adsorption and desorption profiles, irrespective of the validity or otherwise of the pseudo-first-order kinetic approximation. Experimental results obtained for the interaction of histidine-rich glycoprotein with immobilized IgG were then used to illustrate various features of the pseudo-first-order and second-order kinetic analyses, and to determine from the second-order analysis an association equilibrium constant of 2 x 10(8) M-1, which is 20-fold greater than the value obtained by interpretation of the profiles in terms of pseudo-first-order kinetic behavior.

Adsorption↗

PubMed2000

Differential regulation of FGF-1 and -2 mitogenic activity is related to their kinetics of binding to heparan sulfate in MDA-MB-231 human breast cancer cells.

The growth of the malignant human mammary MDA-MB-231 cells is stimulated by fibroblast growth factor-1 (FGF-1) but not by FGF-2. When these cells are cultured in the presence of chlorate, an inhibitor of heparan sulfate (HS) sulfation, their proliferation is stimulated by both FGF-1 and FGF-2. We analyzed the interactions of FGF-1 and FGF-2 with HS purified from the cell layer and the culture medium of control and chlorate-treated MDA-MB-231 cells. The HS from the cell layer bound FGF-1 with faster association kinetics than the HS from the culture medium, and so had a higher affinity for FGF-1. Chlorate treatment had no significant effect on the FGF-1 binding kinetics of the HS. In contrast to FGF-1, chlorate treatment of the cells significantly altered the FGF-2 binding kinetics. The HS from untreated cells possessed two binding sites for FGF-2, one with fast association kinetics (k(ass) 470,000 to 610,000 M(-1) s(-1)) and a high affinity (K(d) 46 to 70 nM) and one with slower association kinetics (k(ass) 74,000 to 100,000 M(-1) s(-1)) and a lower affinity (K(d) 290 to 400 nM). HS from chlorate-treated cells possessed just a single binding site for FGF-2 with fast association kinetics (k(ass) 270,000 to 290,000 M(-1) s(-1)) and a high affinity (K(d) 41 to 57 nM). These results show that there is a relationship between the binding kinetics of FGFs and their ability to stimulate cell growth.

Artifacts↗

PubMedDate not supplied

Kinetic mechanism of DIDS binding to band 3 (AE1) in human erythrocyte membranes.

Stilbenedisulfonates (S) are used widely in cell biology as competitive inhibitors of anion exchange, but the mechanism of competition is not resolved. Resolution requires understanding the detailed steps in the reaction of stilbenedisulfonates with various anion-exchange proteins. Studies on the reversible binding of DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) and H2DIDS (4,4'-diisothiocyanatodihydro-2,2'-stilbenedisulfonate) to erythrocyte band 3 (B) have shown biphasic kinetic time courses at 25 degrees C. Yet, results for the reversible binding of DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) are controversial. One recent report has shown monophasic kinetics, in experiments performed at 0 degrees C, and at a single, very low concentration of DIDS (0.1 microM). Studies are presented which attempt to reconcile these recent findings with the other kinetic data in the literature. We measure the kinetics of DIDS reversible binding to band 3, over a wide DIDS concentration range. In addition, the time course for DIDS binding to band 3 at 0 degrees C is compared with that at 25 degrees C. The results show biphasic binding kinetics at both 0 and 25 degrees C, and they are consistent with expectations for a two-step binding mechanism (S + B <--> SB <--> SB*). Furthermore, computer-assisted model simulation studies reveal that monophasic DIDS binding kinetics are generated by a two-step mechanism, when calculations are performed at 0.1 microM DIDS and 0 degrees C. Under these conditions the initial binding step in the two-step reaction becomes rate limiting. We conclude that the two-step binding mechanism best describes stilbenedisulfonate binding to band 3 and that the observation of monophasic kinetics at low concentrations of DIDS, while valid, is not mechanistically discriminating, since both one-step and two-step mechanisms can yield the same result.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2002

Towards a consistent modeling of protein thermodynamic and kinetic cooperativity: how applicable is the transition state picture to folding and unfolding?

To what extent do general features of folding/unfolding kinetics of small globular proteins follow from their thermodynamic properties? To address this question, we investigate a new simplified protein chain model that embodies a cooperative interplay between local conformational preferences and hydrophobic burial. The present four-helix-bundle 55mer model exhibits protein-like calorimetric two-state cooperativity. It rationalizes native-state hydrogen exchange observations. Our analysis indicates that a coherent, self-consistent physical account of both the thermodynamic and kinetic properties of the model leads naturally to the concept of a native state ensemble that encompasses considerable conformational fluctuations. Such a multiple-conformation native state is seen to involve conformational states similar to those revealed by native-state hydrogen exchange. Many of these conformational states are predicted to lie below native baselines commonly used in interpreting calorimetric data. Folding and unfolding kinetics are studied under a range of intrachain interaction strengths as in experimental chevron plots. Kinetically determined transition midpoints match well with their thermodynamic counterparts. Kinetic relaxations are found to be essentially single-exponential over an extended range of model interaction strengths. This includes the entire unfolding regime and a significant part of a folding regime with a chevron rollover, as has been observed for real proteins that fold with non-two-state kinetics. The transition state picture of protein folding and unfolding is evaluated by comparing thermodynamic free energy profiles with actual kinetic rates. These analyses suggest that some chevron rollovers may arise from an internal frictional effect that increasingly impedes chain motions with more native conditions, rather than being caused by discrete deadtime folding intermediates or shifts of the transition state peak as previously posited.

Calorimetry↗

PubMed1992

Kinetic properties and regulation of GABAA receptor channels.

Single-channel recordings of GABAA receptor single-channel currents have been obtained from mouse spinal cord neurons in cell culture. Detailed kinetic analysis of single-channel main-conductance level currents has allowed development of a preliminary kinetic scheme which describes the gating of the GABAA receptor channel. The essential features of this kinetic scheme are presented in scheme 1 (see above). In this scheme, the GABAA receptor channel is envisioned to exist in multiple open and closed states. Properties can be broken into three main categories. First, the receptor can exist in a closed and nondesensitized set of states. In the kinetic model it is envisioned that there is an unbound (C13), a singly bound (C12), and two doubly bound (C11 and C10) closed states. The singly bound and doubly bound closed states are thought to open to three open states (O1, O2, O3). However, each of the open states opens to two distal closed states whose kinetic properties are similar for all three open states (C4-C9). Only one desensitized state (D0) has been incorporated into the model. While this characterization of desensitization is certainly incomplete, it is an initial step toward including the desensitization process which is clearly evident in whole-cell and single-channel recordings. This kinetic scheme should be considered only an initial working model. A number of features appear to be correct. First, all analyses of open time frequency histograms for GABA- and GABA agonist-induced single-channel openings have demonstrated the presence of at least three distinct open time constants. Furthermore, the concentration-dependent change in the relative frequency of occurrence of the three open states suggests that the open states occur from singly and doubly bound forms of the receptor. Second, the presence of two brief closed states adjacent to the open states appears fairly secure. However, it should be noted that the kinetic analysis primarily suggests that each open state opens to two brief adjacent closed states in a concentration-independent manner. While we have indicated that these two closed states are distal to the three open states, the actual assignment of the location of these states is unclear. Another interpretation of these data is that there is only a single distal closed state and that the proximal, extraburst closed states have very brief durations that are similar to each other. What appears clear, however, is that the open states can close to either brief closed state.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Allosterism and Na(+)-D-glucose cotransport kinetics in rabbit jejunal vesicles: compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved.

We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na(+)-D-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na+ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na+: 1 S and 1 Na+: 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na(+)-D-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na+ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na+ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na+ electrochemical gradient to the transporter.

Allosteric Regulation↗

PubMed1984

Voltage-dependent kinetics of an anionic channel of large unit conductance in macrophages and myotube membranes.

Using the patch-clamp technique single-channel parameters and kinetic properties of an anionic channel are studied in cell-attached and excised membrane patches from peritoneal macrophages of mouse and cultured chicken myotubes. The channel has a unit conductance of about 340 pS with a Q10 of 1.3. In addition a subconductance state of about 210 pS is frequently adopted. The selectivity ratio of PCl/PNa is about 5. In excised membrane patches the activation of the channel appears to be independent of Ca either in the cytoplasmic or the extracellular medium. The channel induced current fluctuations appear in a burst like pattern. At least three non-conducting channel states could be distinguished kinetically. The mean lifetime of one of these states exhibits a strikingly steep voltage dependence which could be correlated to the mean shut interval between consecutive bursts. A similar steep voltage dependence was found for the mean lifetimes of bursts. The burst kinetic shows an about bell-shaped dependence on voltage. The results suggest that the burst kinetic and the kinetic within bursts are regulated by independent voltage sensitive mechanisms. The burst kinetic was analyzed by ensemble averages of voltage-jump current relaxations performed on the single channel level. A model of two voltage-sensitive gates is proposed for a description of the burst kinetic.

Animals↗

PubMed1991

Kinetic comparison of caiman epsilon-crystallin and authentic lactate dehydrogenases of vertebrates.

Kinetic comparison of epsilon-crystallins isolated from the avian and reptilian species and the authentic lactate dehydrogenases (LDHs) was undertaken in order to clarify the identities of these structural lens proteins in relation to their enzymatic activity. Caiman epsilon-crystallin similar to the previously characterized duck epsilon-crystallin appeared to possess a genuine and stable LDH activity as detected by nitro blue tetrazolium staining on polyacrylamide gels and conventional kinetic assays. Kinetic parameters for pyruvate, L-lactate, NAD+, and three structural analogues of the coenzyme in this epsilon-crystallin catalyzed reaction were also determined and compared. Despite the structural similarities between epsilon-crystallins and chicken heart LDH, differences in charge and kinetic properties have been revealed by native isozyme electrophoresis and kinetic analysis as examined by initial velocity and substrate inhibition studies. It is found that the kinetic data analyzed for caiman epsilon-crystallin were more fitted with a compulsory ordered Bi-Bi sequential mechanism similar to those for the authentic LDHs and duck epsilon-crystallin. Caiman epsilon-crystallin has for the first time been established as a heart-type LDH based on the kinetic analysis and comparison with the authentic heart- and muscle-type LDHs from pig and chicken.

Alligators and Crocodiles↗

PubMed1996

Effect of intravascular ligand binding on parameter estimates derived from tracer kinetic modelling.

The purpose of this study was to assess the effect of intravascular ligand binding on parameter estimates derived from tracer kinetic modelling. To this end intravascular ligand kinetics between the free and a bound compartment in plasma and the exchange of tracer between the capillary space and tissue were analysed using a simple compartment model. The effect of non-equilibrated intravascular compartments on parameter estimates was evaluated in a computer simulation. It was found that three kinetic situations must be distinguished. If the intravascular compartments are fully equilibrated when the ligand reaches the target organ, intravascular binding simply acts as a scale factor for the transport-related parameter K1. If the intravascular kinetics is very slow, only minimal binding will occur. In between there is a range where ongoing equilibration leads to time variability of K1. Since tracer kinetic modelling usually does not account for such time variability, the parameter estimates become biased, the degree of the bias depending on the intravascular binding kinetics. Furthermore the bias may be dependent on receptor density, meaning that model-derived receptor estimates are not linearly related to the true receptor density. It is concluded that intravascular ligand binding can severely affect parameter estimates derived from tracer kinetic modelling. Especially disturbing are effects due to ongoing intravascular equilibration following the arrival of the ligand in the target organ. These can be avoided by letting the ligand equilibrate with blood in a syringe prior to injection.

Blood Vessels↗

PubMedDate not supplied

An internet-based "kinetic imaging system" (KIS) for MicroPET.

Many considerations, involving understanding and selection of multiple experimental parameters, are required to perform MicroPET studies properly. The large number of these parameters/variables and their complicated interdependence make their optimal choice nontrivial. We have a developed kinetic imaging system (KIS), an integrated software system, to assist the planning, design, and data analysis of MicroPET studies. The system serves multiple functions-education, virtual experimentation, experimental design, and image analysis of simulated/experimental data-and consists of four main functional modules--"Dictionary," "Virtual Experimentation," "Image Analysis," and "Model Fitting." The "Dictionary" module provides didactic information on tracer kinetics, pharmacokinetic, MicroPET imaging, and relevant biological/pharmacological information. The "Virtual Experimentation" module allows users to examine via computer simulations the effect of biochemical/pharmacokinetic parameters on tissue tracer kinetics. It generates dynamic MicroPET images based on the user's assignment of kinetics or kinetic parameters to different tissue organs in a 3-D digital mouse phantom. Experimental parameters can be adjusted to investigate the design options of a MicroPET experiment. The "Image Analysis" module is a full-fledged image display/manipulation program. The "Model Fitting" module provides model-fitting capability for measured/simulated tissue kinetics. The system can be run either through the Web or as a stand-alone process. With KIS, radiotracer characteristics, administration method, dose level, imaging sequence, and image resolution-to-noise tradeoff can be evaluated using virtual experimentation. KIS is designed for biology/pharmaceutical scientists to make learning and applying tracer kinetics fun and easy.

Animals↗

PubMed1983

Kinetic compartmental analysis of carnitine metabolism in the dog.

This study was undertaken to quantitate the dynamic parameters of carnitine metabolism in the dog. Six mongrel dogs were given intravenous injections of L-[methyl-3H]carnitine and the specific radioactivity of carnitine was followed in plasma and urine for 19-28 days. The data were analyzed by kinetic compartmental analysis. A three-compartment, open-system model [(a) extracellular fluid, (b) cardiac and skeletal muscle, (c) other tissues, particularly liver and kidney] was adopted and kinetic parameters (carnitine flux, pool sizes, kinetic constants) were derived. In four of six dogs the size of the muscle carnitine pool obtained by kinetic compartmental analysis agreed (+/- 5%) with estimates based on measurement of carnitine concentrations in different muscles. In three of six dogs carnitine excretion rates derived from kinetic compartmental analysis agreed (+/- 9%) with experimentally measured values, but in three dogs the rates by kinetic compartmental analysis were significantly higher than the corresponding rates measured directly. Appropriate chromatographic analyses revealed no radioactive metabolites in muscle or urine of any of the dogs. Turnover times for carnitine were (mean +/- SEM): 0.44 +/- 0.05 h for extracellular fluid, 232 +/- 22 h for muscle, and 7.9 +/- 1.1 h for other tissues. The estimated flux of carnitine in muscle was 210 pmol/min/g of tissue. Whole-body turnover time for carnitine was 62.9 +/- 5.6 days (mean +/- SEM). Estimated carnitine biosynthesis ranged from 2.9 to 28 mumol/kg body wt/day. Results of this study indicate that kinetic compartmental analysis may be applicable to study of human carnitine metabolism.

Animals↗

PubMed1992

The pH dependence of the kinetic parameters of ketol acid reductoisomerase indicates a proton shuttle mechanism for alkyl migration.

The enzyme ketol acid reductoisomerase catalyzes the second common reaction in the biosynthesis of the branched chain amino acids. The reaction is complex as an alkyl migration and a ketone reduction apparently occur as separate steps during the conversion of acetolactate to 2,3-dihydroxy-3-methylbutyrate. This paper reports on the pH dependence of the kinetic parameters of the enzyme. The pH variation of log(V/K) for acetolactate was fit to an equation describing a bell-shaped curve, indicating an acid and a base catalyst for the reaction. In the reverse direction, V/K for 2,3-dihydroxy-3-methylbutyrate is constant over the pH range 8 to 10 and decreases below pH 8 with the ionization of two catalytic groups. The pH dependence of the V/K values for reduction of the kinetically competent intermediate and analogs of this intermediate are also described by a bell-shaped curve. The pH dependence of the V/K for alkyl migration of this intermediate indicates a single base catalyst for this reaction. We observe no deuterium kinetic isotope effect on V or V/K for the reaction of acetolactate at any pH. We observe a pH-dependent kinetic isotope effect on V/K for the reduction of the intermediate, the magnitude of which is metal ion dependent. Larger KIE's are observed in the presence of Mn2+ as opposed to Mg2+. In the reverse reaction there is a pH-dependent kinetic isotope effect on V/K. Based on the pH dependence of the kinetic parameters and the kinetic isotope effects, we propose a base-catalyzed proton shuttle mechanism for the alkyl migration reaction followed by an acid-assisted ketone reduction by NADPH.

Alcohol Oxidoreductases↗

PubMed1994

Effects of cocaine and repeated cocaine followed by withdrawal. Alterations of dopaminergic transporter turnover with no changes in kinetics of substrate recognition.

The turnover of the transporter for dopamine (ca. 1.5 sec-1) and the apparent second order rate constant of association of dopamine with the outward facing form of the transporter protein (ca. 10(6) M-1sec-1) were estimated using kinetic data from rotating disk voltammetric measures of the inward transport of dopamine in striatal suspensions, standard treatments of the kinetics of transport, and values in the literature for density of striatal transporter sites. Under apparent steady-state conditions of transporter functioning, inhibition of the transport of dopamine by cocaine following its addition to the incubation buffer was found to decrease the turnover of the transporter and not affect the kinetics of substrate recognition. The kinetics of binding of dopamine to the transporter were estimated also by apparent pre-steady-state kinetics. These experiments confirmed the second order nature of the binding of dopamine to the transporter and the numerical value of the rate constant estimated under steady-state conditions; they also demonstrated that the binding of dopamine has an absolute dependence on Na+, and that the second order rate constant of association of dopamine with its transporter is not influenced by cocaine. In separate studies, similar experiments were conducted in tissues from animals that had been treated with cocaine for 3 days and withdrawn for 1 day or 2 weeks. Repeated treatments with cocaine followed by either a 24-hr or 2-week period of withdrawal resulted in increases in the Vmax and turnover of the transporter with no apparent changes in the kinetics of association of substrate. No differences between the Ki for cocaine observed in direct inhibitions of the transport of dopamine and the Ki for cocaine observed in tissues obtained from animals treated repeatedly with cocaine were observed. Taken together, these data suggest that cocaine exerts its effects by altering an intramembrane translocation step for the movement of dopamine and not by changing the recognition of dopamine by the externally facing binding site or the apparent Ki for cocaine. Finally, repeated treatments with cocaine followed by a period of withdrawal appear to kinetically activate the transporter for dopamine.

Animals↗

PubMed1995

Comparison of the potency, kinetics and voltage-dependency of a series of uncompetitive NMDA receptor antagonists in vitro with anticonvulsive and motor impairment activity in vivo.

The amino-adamantane derivatives memantine (1-amino-3,5-dimethyladamantane) and amantadine (1-amino-adamantane) are relatively low affinity, uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists which have been used clinically in the treatment of dementia and Parkinson's disease respectively for several years without serious side effects. The aim of this study was to test whether memantine, amantadine and other low affinity uncompetitive NMDA receptor antagonists also have better therapeutic indices than high affinity antagonists in preclinical models of epilepsy by assessing the potency, kinetics and voltage-dependency of open channel blockade for a series antagonists in vitro and comparing these effects to anticonvulsive and motor impairment activity in vivo. The compounds tested were memantine, amantadine, 14 other amino-adamantanes, (+)-MK-801, ketamine, dextrorphan, dextromethorphan and phencyclidine. The offset kinetics of open-channel blockade assessed with whole cell patch clamp recordings from cultured superior colliculus neurones were highly correlated to potency i.e. the less potent antagonists showed faster unblocking kinetics (Koff, r = 0.904). Although, onset kinetics as assessed by Kon were not correlated to potency (r = 0.023), tau on estimated at IC50 is perhaps a more meaningful measure of onset kinetics at equieffective concentrations and was also well correlated to potency (r = -0.863). All amino-adamantanes tested were strongly voltage-dependent. There was also a good correlation between the in vitro potencies of uncompetitive NMDA receptor antagonists assessed with patch clamp recordings and displacement of equilibrium [3H](+)-MK-801 binding and their in vivo activity against maximal electroshock (MES) and pentylenetetrazol (PTZ) induced tonic convulsions and NMDA-induced lethality in mice. Memantine and four other amino-adamantanes with somewhat lower potency and faster blocking kinetics had better therapeutic indices (ED50 rotarod and traction reflex over ED50 in MES-induced convulsions; TI = 2-4) than substances with higher affinity such as ketamine, dextrorphan and (+)-MK-801 (TI < 2). However, amantadine and several other amino-adamantanes with lower potency than memantine actually had poorer therapeutic indices (TI < or = 0.5) which may have been due to additional actions at other ion channels or receptors at the doses necessary to protect against seizures. In fact, ED50 in the MES test was negatively-correlated to therapeutic indices (traction r = -0.790, rotarod r = -0.797) i.e. the less potent uncompetitive antagonists had worse therapeutic indices. The data from the present study do not lend support to the idea that low affinity, open channel NMDA receptor blockers are also effective in models of epilepsy at doses having little effect on physiological processes. It should be stressed that these data do not contradict the known therapeutic safety of memantine and amantadine in dementia and Parkinson's disease respectively. Thus the good clinical profile of memantine in dementia has been attributed not only to its fast blocking/unblocking kinetics but also to its strong voltage-dependency. These biophysical properties may allow therapeutically-relevant concentrations to block chronic, low level pathological activation of NMDA receptors whilst leaving their synaptic activation intact. Precisely these properties may also underlie the poor therapeutic indices seen in the present study on antiepileptic activity due to the synaptic nature of both seizures and normal glutamatergic transmission.

Amantadine↗

PubMed1996

Carbon dioxide recovery in ready biodegradation tests: mass transfer and kinetic considerations.

The kinetics and efficiency of carbon dioxide recovery in modern versions of the Sturm Ready Biodegradation Test were examined to determine the ability of CO2 evolution measurements to accurately estimate the rate and extent of ultimate biodegradation (mineralization). Kinetic data were analyzed by nonlinear regression techniques using an automated curve-fitting package available from commercial sources. The kinetics of CO2 recovery in standard 3.8 L glass carboys containing 2 L of medium were rapid when headspace aeration (approximately 6 ml/min) and moderate agitation (140 rev/min) on a rotary platform shaker were used to ensure adequate aeration and mixing. The time (half-life) for 50% CO2 recovery in external base traps was 4-5 hours, and stoichiometric recoveries of CO2 equivalents added as bicarbonate were obtained within 24 hours. The kinetics of CO2 evolution during biodegradation of several test compounds were significantly slower than the kinetics of CO2 recovery, with half-lives between 65 and 191 hours. Our results indicate that mass transfer limitations do not impact CO2 recoveries or biodegradation kinetic measurements in modern versions of the Sturm Test, even in test vessels with relatively low surface area to volume ratios (1:1). The use of headspace aeration and mixing generates reliable kinetic data, which can be analyzed by commercially-available nonlinear regression packages to provide rate information for the classification of chemicals with different biodegradation profiles.

Biodegradation, Environmental↗

PubMed1989

Modelling of chemical reactions catalysed by membrane-bound enzymes. Determination and significance of the kinetic constants.

A model of multiphasic systems, based on the assumption of zero-order partition of substrates and products into the membranes, is applied to reversible mono-substrate and bi-substrate reactions catalysed by membrane-bound enzymes. Apart from replacement of single-phase kinetic constants by apparent kinetic constants, the derived kinetic expressions are formally identical with those for corresponding single-phase systems. The model confers to the apparent kinetic constants an experimentally verifiable meaning. For full characterization of membrane-kinetic systems, experiments at various concentrations of enzyme-embedding phospholipid are required. Extrapolation to zero phospholipid concentration of each Km app then yields the corresponding true kinetic constant characteristic of the membrane-bound enzyme and also provides a technique for determination of the membrane-partition constants. The procedure implies that the phospholipid content should be assayed for full characterization of membrane-bound enzymes. If, for practical reasons, the assays have to be limited to a single enzyme concentration, correction of the apparent kinetic constants is still possible provided the phospholipid concentration and the partition constants of the reactants are known. The model has permitted prediction of a number of previous observations reflecting the multiphasic nature of the systems. The assumptions, underlying the model, and their implications are examined as well as some commonly used experimental designs for determination of the type of enzymic site.

Binding Sites↗

PubMed2006

Why substrate depletion has apparent first-order kinetics in enzymatic digestion.

A number of enzyme digestion assays show apparent first-order kinetics of reactant depletion. There are four possible explanations of this phenomenon: (i) the reaction is dominated by a first-order limiting step, (ii) the digestion follows a pseudo-first-order kinetics under the excess of a reactant species, (iii) the first-order kinetics is only applicable to the slow transient of the reaction, or (iv) the aggregate behavior of the reaction pathway produces behavior indistinguishable from the first-order kinetics. In this paper, we investigate the kinetics for protein digestion by formulating rate equations for two proposed mechanisms, namely the one-by-one mechanism and the zipper mechanism. Our analysis shows that the kinetics of protein digestion follows apparent first-order kinetics irrespective of the mechanism for low initial substrate concentration compared to the initial enzyme concentration. Also, our results provide an explanation for experimental observations and suggest a new experimental protocol that could reveal information on the mechanism of digestion.

Digestion↗

PubMed2003

Surface-induced dissociation of peptide ions: kinetics and dynamics.

Kinetics and dynamics studies have been carried out for the surface-induced dissociation (SID) of a set of model peptides utilizing a specially designed electrospray ionization Fourier Transform ion cyclotron resonance mass spectrometer in which mass-selected and vibrationally relaxed ions are collided on a orthogonally-mounted fluorinated self-assembled monolayer on Au [111] crystal. The sampling time in this apparatus can be varied from hundreds of microseconds to tens of seconds, enabling the investigation of kinetics of ion decomposition over an extended range of decomposition rates. RRKM-based modeling of these reactions for a set of polyalanines demonstrates that SID kinetics of these simple peptides is very similar to slow, multiple-collision activation and that the distribution of internal energies following collisional activation is indistinguishable from a thermal distribution. For more complex peptides comprised of several amino acids and with internal degrees of freedom (DOF) of the order of 350 there is a dramatic change in kinetics in which RRKM kinetics is no longer capable of describing the decomposition of these complex ions. A combination of RRKM kinetics and the "sudden death" approximation, according to which decomposition occurs instantaneously, is a satisfactory description. This implies that a population of ions-which is dependant on the nature of the peptide, kinetic energy and sampling time-decomposes on or very near the surface. The shattering transition is described quantitatively for the limited set of molecules examined to date.

Cyclotrons↗