Dynamics of myo1c (myosin-ibeta ) lipid binding and dissociation

Myosin-I is the single-headed member of the myosin superfamily that associates with lipid membranes. Biochemical experiments have shown that myosin-I membrane binding is the result of electrostatic interactions between the basic tail domain and acidic phospholipids. To better understand the dynamics of myosin-I membrane association, we measured the rates of association and dissociation of a recombinant myo1c tail domain (which includes three IQ domains and bound calmodulins) to and from large unilamellar vesicles using fluorescence resonance energy transfer. The apparent second-order rate constant for lipid-tail association in the absence of calcium is fast with nearly every lipid-tail collision resulting in binding. The rate of binding is decreased in the presence of calcium. Time courses of myo1c-tail dissociation are best fit by two exponential rates: a fast component that has a rate that depends on the ratio of acidic phospholipid to myo1c-tail (phosphatidylserine (PS)/tail) and a slow component that predominates at high PS/tail ratios. The dissociation rate of the slow component is slower than the myo1c ATPase rate, suggesting that myo1c is able to stay associated with the lipid membrane during multiple catalytic cycles of the motor. Calcium significantly increases the lifetimes of the membrane-bound state, resulting in dissociation rates 0.001 s(-1).     

Folding and assembly of dimeric human glutathione transferase A1-1

Glutathione transferases function as detoxification enzymes and ligand-binding proteins for many hydrophobic endogenous and xenobiotic compounds. The molecular mechanism of folding of urea-denatured homodimeric human glutathione transferase A1-1 (hGSTA1-1) was investigated. The kinetics of change were investigated using far-UV CD, Trp20 fluorescence, fluorescence-detected ANS binding, acrylamide quenching of Trp20 fluorescence, and catalytic reactivation. The very early stages of refolding (millisecond time range) involve the formation of structured monomers with native-like secondary structure and exposed hydrophobic surfaces that have a high binding capacity for the amphipathic dye ANS. Dimerization of the monomeric intermediates was detected using Trp fluorescence and occurs as fast and intermediate events. The intermediate event was distinguished from the fast event because it is limited by a preceding slow trans-to-cis isomerization reaction (optically silent in this study). At high concentrations of hFKBP, dimerization is not limited by the isomerization reaction, and only the fast event was detected. The fast (tau = 200 ms) and intermediate (tau = 2.5 s) events show similar urea-, temperature-, and ionic strength-dependent properties. The dimeric intermediate has a partially functional active site ( approximately 20%). Final reorganization to form the native tertiary and quaternary structures occurs during a slow, unimolecular, urea- and ionic strength-independent event. During this slow event (tau = 250 s), structural rearrangements at the domain interface occur at/near Trp20 and result in burial of Trp20. The slow event results in the regain of the fully functional dimer. The role of the C-terminus helix 9 (residues 210-221) as a structural determinant for this final event is proposed.     

Static and kinetic studies of complex formations between calmodulin and mastoparanX

Ca(2+)-induced complex formation between calmodulin (CaM) and mastoparanX (MasX) was studied by a fluorescence spectroscopy and by a stopped-flow method. The measurements of the fluorescence anisotropy in the presence of calcium and the fluorescence titration with Ca(2+) revealed that the N- and C-domains of CaM bound cooperatively MasX, while the tryptic fragments of CaM (TR(1)C, 1-77 and TR(2)C, 78-148) bound independently MasX. The Trp-fluorescence stopped-flow experiments revealed that the Ca(2+)-induced binding of CaM and MasX was composed of two processes: one was a rapid binding of the N-domain of CaM to MasX, which was induced by the rapid Ca(2+) binding to the N-sites of CaM. The other was a slow biphasic process. Its fast phase was the binding of the C-domain of CaM to MasX, which was induced by the slow Ca(2+) binding to the C-sites. Interestingly, the kinetics of the slow process varied with the Ca(2+) concentrations. At the low Ca(2+) concentrations, its rate constant increased to around 20 s(-1) as the Ca(2+) concentration increased. At the high Ca(2+) concentrations, the Ca(2+)-induced binding of the C-domain of CaM to MasX proceeded at a constant rate around 20 s(-1). This suggested an existence of a rate-limiting step for the Ca(2+)-induced binding of the C-domain of CaM to MasX at the high Ca(2+) concentrations. The slow phase of the slow process may be a rearrangement of the CaM-MasX complex. These results led to our model of a molecular kinetic mechanism of the Ca(2+)-induced complex formation between CaM and MasX.     

Kinetics of binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The mechanism of binding between thermolysin with its specific inhibitor, talopeptin (MKI), was studied kinetically with the stopped-flow method by monitoring the enhancement of tryptophan fluorescence caused by the complex formation. Only one relaxation obeying first-order kinetics was observed. The dependence of the apparent first-order constant, kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular binding step followed by a slow unimolecular step. It was found that the increase in tryptophan fluorescence occurs solely in the slow unimolecular step. The apparent second-order rate constant, (kon)app, in the low inhibitor concentration range, was determined over the pH range between 5 and 8.5 and decreases with increasing pH. The activation parameters for the overall binding process were obtained from the temperature dependence of (kon)app.     

pH dependence of folding of iso-2-cytochrome c

Starting from a standard unfolded state (3.0 M guanidine hydrochloride, pH 7.2), the kinetics of refolding of iso-2-cytochrome c have been investigated as a function of final pH between pH 3 and pH 10. Absorbance in the ultraviolet and visible spectral regions and tryptophan fluorescence are used to monitor folding. Over most of the pH range, fast and slow folding phases are detected by both fluorescence and absorbance probes. Near neutral pH, the rate of fast folding appears to be the same when monitored by absorbance and fluorescence probes. At higher and lower pH, there are two fast folding reactions, with absorbance-detected fast folding occurring in a slightly faster time range than fluorescence-detected fast folding. The rates of both fast folding reactions pass through broad minima near neutral pH, indicating involvement of ionizable groups in rate-limiting steps. The rates of slow folding also depend on the final pH. At acid pH, there appears to be a single slow folding phase for both fluorescence and absorbance probes. At neutral pH, the absorbance-detected and fluorescence-detected slow folding phases separate into distinct kinetic processes which differ in rate and relative amplitude. At high pH, absorbance-detected slow folding is no longer observed, while fluorescence-detected slow folding is decreased in amplitude. In contrast, the equilibrium and kinetic properties of proline imide bond isomerization, believed to be involved in the slow folding reactions, are largely independent of pH. The results suggest that the pH dependence of slow folding involves coupling of pH-sensitive structure to proline imide bond isomerization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of melittin binding to phospholipid small unilamellar vesicles

We have used the decrease in the fluorescence intensity of the single tryptophan residue of bee venom melittin at long emission wavelengths that accompanies binding of the peptide to phospholipid small unilamellar vesicles to determine the rate of binding through the use of stopped-flow fluorometry in the millisecond range. We have found the rate to depend on the degree of saturation of the lipid acyl chains as well as on the physical state of the bilayer, the net electric charge of the polar headgroups, and the lipid-to-melittin molar ratio R. For zwitterionic lipids (i) the binding process is found to exhibit negative cooperativity, and (ii) the rate-limiting step appears to be penetration of the protein into the hydrophobic region of the bilayer. For negatively charged lipids the results show that binding is a very fast process that seems to be electrostatic in nature.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

Formation and movement of Fe(III) in horse spleen, H- and L-recombinant ferritins. A fluorescence study.

Iron oxidation and incorporation into apoferritins of different subunit composition, namely the recombinant H and L homopolymers and the natural horse spleen heteropolymer (10-15% H), have been followed by steady-state and time-resolved fluorescence. After aerobic addition of 100 Fe(II) atoms/polymer, markedly different kinetic profiles are observed. In the rL-homopolymer a slow monotonic fluorescence quenching is observed which reflects binding, slow oxidation at the threefold apoferritin channels, and diffusion into the protein cavity. In the rH-homopolymer a fast fluorescence quenching is followed by a partial, slow recovery. The two processes have been attributed to Fe(II) binding and oxidation at the ferroxidase centers and to Fe(III) released into the cavity, respectively. The fluorescence kinetics of horse spleen apoferritin is dominated by the H chain contribution and resembles that of the H homopolymer. It brings out clearly that the rate of the overall process is limited by the rate at which Fe(III) leaves the ferroxidase centers of the H chains where binding of incoming Fe(II) and its oxidation take place. The data obtained upon stepwise addition of iron and the results of optical absorption measurements confirm this picture. The correspondence between steady-state and time-resolved data is remarkably good; this is manifest when the latter are used to calculate the change in fluorescence intensity as apparent in the steady-state measurements.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: V. Qualitative correlation between pharmacological effects and equilibration processes of the cholinergic receptor protein as revealed by the structural probe quinacrine

When the differential fluorescence emission at 515 nm of receptor-rich membrane fragments from Torpedo marmorata labelled with quinacrine is followed by energy transfer, addition of a high concentration of an agonist such as 0.4 mm-carbamylcholine or 0.4 mm-phenyltrimethylammonium causes a fast (unresolved) increase of fluorescence intensity followed by a slow (minute range) decrease, which leads to a final stable level. On the other hand, a stepwise addition of agonist at low concentrations gives only a slow fluorescence increase. Antagonists, such as flaxedil and d-tubocurarine, at all the concentrations tested, bring about exclusively slow fluorescence increases. Decamethonium at 0.4 mm gives no slow reaction but only a fast (unresolved) increase without transient overshoot.Addition of a local anaesthetic reduces the amplitude of the fluorescence response to carbamylcholine. The α-toxin from Naja nigricollis does not cause any change of fluorescence intensity but blocks the effect of the cholinergic agonists and antagonists.Dissolution of the membrane fragments by a non-ionic detergent abolishes the fast transient reaction triggered by the agonists but preserves the slow ones observed in the presence of agonists or antagonists. The data are interpreted in terms of a three-state model, a revised version of the model of Katz & Thesleff (1957): the fast transient reaction brought about by the agonists being assigned to the “activation” of the receptor-ionophore complex and the slow one to its “desensitization”.     

Interaction between DMPC liposomes and HM-PNIPAM polymer

The interactions of hydrophobically-modified poly-(N-isopropylacrylamides) (HM-PNIPAM) and dimyristoylphosphatidylcholine (DMPC) vesicles were investigated by the effect of the polymer on the binding of a fluorescent dye, oxonol VI, to DMPC vesicles, and on its diffusion across the membrane. On mixing with the vesicles, the dye exhibits an increase in fluorescence, which occurs in a two-stage process. The process was monitored by stopped-flow fluorescence spectrophotometry. According to the dependence of the reciprocal relaxation time on vesicle concentration, the rapid stage seems to be due to the second-order binding of the dye to the lipid membrane, a process that is almost diffusion-controlled, whereas the slow process is attributed to movement of the dye within the membrane phase. The polymer did not significantly affect the rate constant of the binding step, but it slowed down slightly the dissociation process of the dye from the membrane. However, the polymer affected the second stage, causing an increase in the reciprocal of its relaxation time, which suggests that the polymer makes the vesicle membrane more fluid.     

Kinetics of anesthetic-induced conformational transitions in a four-alpha-helix bundle protein

Inhaled anesthetics are thought to alter the conformational states of Cys-loop ligand-gated ion channels (LGICs) by binding within discrete cavities that are lined by portions of four alpha-helical transmembrane domains. Because Cys-loop LGICs are complex molecules that are notoriously difficult to express and purify, scaled-down models have been used to better understand the basic molecular mechanisms of anesthetic action. In this study, stopped-flow fluorescence spectroscopy was used to define the kinetics with which inhaled anesthetics interact with (Aalpha(2)-L1M/L38M)(2), a four-alpha-helix bundle protein that was designed to model anesthetic binding sites on Cys-loop LGICs. Stopped-flow fluorescence traces obtained upon mixing (Aalpha(2)-L1M/L38M)(2) with halothane revealed immediate, fast, and slow components of quenching. The immediate component, which occurred within the mixing time of the spectrofluorimeter, was attributed to direct quenching of tryptophan fluorescence upon halothane binding to (Aalpha(2)-L1M/L38M)(2). This was followed by a biexponential fluorescence decay containing fast and slow components, reflecting anesthetic-induced conformational transitions. Fluorescence traces obtained in studies using sevoflurane, isoflurane, and desflurane, which poorly quench tryptophan fluorescence, did not contain the immediate component. However, these anesthetics did produce the fast and slow components, indicating that they also alter the conformation of (Aalpha(2)-L1M/L38M)(2). Cyclopropane, an anesthetic that acts with unusually low potency on Cys-loop LGICs, acted with low apparent potency on (Aalpha(2)-L1M/L38M)(2). These results suggest that four-alpha-helix bundle proteins may be useful models of in vivo sites of action that allow the use of a wide range of techniques to better understand how anesthetic binding leads to changes in protein structure and function.     

Enzymatic activities and ATP-induced fluorescence enhancement of myosin from fast and slow skeletal and cardiac muscles.

The maximal ATP-induced enhancement of fluorescence and the dependence of this enhancement on ATP concentration were determined for myosins from fast and slow skeletal and cardiac muscle of the rabbit. With myosins from slow and cardiac muscle modifications in the preparative procedure and chromatography on DEAE-Sephadex were required to obtain preprations which were free of actin, which exhibited the maximal fluorescence enhancement and which bound two moles of ATP per mole of myosin. Since the fluorescence enhancement of cardiac and slow muscle myosins is labile at slightly alkaline pH, it was also necessary to minimize incubation at pH greater than 7 in order to attain the maximal enhancement. With fast muscle myosin the changes in preparative procedure, together with chromatography, led to a 50 to 100% increase in the steady-state rate of ATP hydrolysis and fluorescence enhancement, without changing the maximal binding of ATP. From a comparison of the rate of steady-state hydrolysis of ATP with the rate of decay of the enhanced fluorescence, it appears that for all three myosins, both ATP binding sites have the same enzymatic activity, the steady-state rate per site being slower for cardiac and slow muscle myosins than for fast muscle myosin.     

Interaction of zinc protoporphyrin with intact oxyhemoglobin

In erythropoietic protoporphyria and lead poisoning, free protoporphyrin (PPIX) and zinc protoporphyrin (ZPP), respectively, accumulate in erythrocytes. That PPIX and ZPP bind to human hemoglobin A (Hb4) is established, but the site of binding is still a matter of controversy. We investigated the interaction of ZPP with intact, tetrameric oxy Hb4, using batch microcalorimetry, front-face fluorometry, absorption difference spectroscopy, oxygen equilibrium studies, and isoelectric focusing (IEF). In the presence of oxy Hb4 (pH 7.35, 0.05 M phosphate), the fluorescence emission maximum (excitation at 420 nm) of ZPP immediately shifts from 587 nm (ZPP alone) to 594 nm, as expected when binding to protein. The fluorescence intensity increases with time and is correlated with the ZPP:Hb4 mole ratio. A slow, time-dependent reaction is also observed with microcalorimetry: the rate of heat of reaction exhibits both a fast and a slow component. The heats of reaction range from -2.1 to -14.8 mcal depending upon the ZPP:Hb4 ratio of 4:1 (0.4 mM:0.1 mM) to 38:1 (3.8 mM:0.1 mM), respectively, and are typical of weak, noncovalent protein-ligand interactions. The optical difference spectra are a function of the ZPP:Hb4 molar ratio and also exhibit a slow increase in intensity over time. No time-dependent optical difference spectra are observed with ZPP or with Hb4 alone. The oxygen affinity of Hb4 in the presence of ZPP decreases with increasing mole ratio. During IEF, all ZPP separates from Hb4, consistent with a weak, noncovalent interaction at a non-heme pocket site. We conclude that ZPP binds to intact, tetrameric hemoglobin at non-heme pocket sites in a nonspecific, weak, noncovalent interaction.     

GTP hydrolysis mechanisms in ras p21 and in the ras-GAP complex studied by fluorescence measurements on tryptophan mutants

We have substituted leucine 56 or tyrosine 64 of p21 ras with a tryptophan. The intrinsic fluorescence of this tryptophan was used as an internal conformational probe for time-resolved biochemical studies of the ras protein. The slow intrinsic GTPase, GDP/GTP exchange induced by the SDC25 "exchange factor", and the fast GTP hydrolysis induced by GAP were studied. Tryptophan fluorescence of mutated ras is very sensitive to magnesium binding, GDP/GTP exchange, and GTP hydrolysis (changes in tyrosine fluorescence of wild-type ras are also observed but with a lower sensitivity). Nucleotide affinities, exchange kinetics, and intrinsic GTPase rates of the mutated ras could be measured by this method and were found to be close to those of wild-type ras. The SDC25 gene product enhances GDP/GTP exchange in both mutants. In both mutants, a slow fluorescence change follows the binding of GTP gamma S; its kinetics are close to those of the intrinsic GTPase, suggesting that a slow conformational change precedes the GTPase and is the rate-limiting step, as proposed by Neal et al. (1990) (Proc. Natl. Acad. Sci. U.S.A. 87, 3562-3565). GAP interacts with both mutant ras proteins and accelerates the GTPase of (L56W)ras but not that of (Y64W)ras, suggesting a role for tyrosine 64 in GAP-induced GTP hydrolysis. However, GAP does not accelerate the slow conformational change following GTP gamma S binding in either of the mutated ras proteins. This suggests that the fast GAP-induced catalysis of GTP hydrolysis that is observed with (L56W)ras bypasses the slow conformational change associated with the intrinsic GTPase and therefore might proceed by a different mechanism.     

Effects of deferrioxamine on iron-catalyzed lipid peroxidation.

The kinetics of iron binding by deferrioxamine B mesylate and the ramifications of this process upon iron-catalyzed lipid peroxidation were assessed. The relative rates of Fe(III) binding by deferrioxamine varied for the chelators tested as follows: ADP greater than AMP greater than citrate greater than histidine greater than EDTA. The addition of a fivefold molar excess of deferrioxamine to that of Fe(III) did not result in complete binding (within 10 min) for any of the Fe(III) chelates tested except ADP:Fe(III). The rates of Fe(III) binding by deferrioxamine were greater at lower pH and when the competing chelator concentration was high in relationship to iron. The relatively slow binding of Fe(III) by deferrioxamine also affected lipid peroxidation, an iron-dependent process. The addition of deferrioxamine to an ascorbate- and ADP:Fe(III)-dependent lipid peroxidation system resulted in a time-dependent inhibition or stimulation of malondialdehyde formation (i.e., lipid peroxidation), depending on the ratio of deferrioxamine to iron. Converse to Fe(III), the rates of Fe(II) binding by deferrioxamine from the chelators tested above were rapid and complete (within 1 min), and resulted in the oxidation of Fe(II) to Fe(III). Lipid peroxidation dependent on Fe(II) autoxidation was stimulated by the addition of deferrioxamine. Malondialdehyde formation in this system was inhibited by the addition of catalase, and a similar extent of lipid peroxidation was achieved by substituting hydrogen peroxide for deferrioxamine. Collectively, these results suggest that the kinetics of Fe(III) binding by deferrioxamine is a slow, variable process, whereas Fe(II) binding is considerably faster. The binding of either valence of iron by deferrioxamine may result in variable effects on iron-catalyzed processes, such as lipid peroxidation, either via slow binding of Fe(III) or the rapid binding of Fe(II) with concomitant Fe(II) oxidation.     

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers

Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non-Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.     

Expanding the scope of quantitative FRAP analysis

In this study, new mathematical models were developed for analysis of fluorescence recovery after photobleaching (FRAP) data to account for features not represented in previous analysis: conical photobleaching geometry, spatial variations in binding of fluorescent molecules, and directed transport of fluorescent molecules. To facilitate computations in conical geometry, a fast computational method for calculation of fluorescence recovery is presented. Two approximations are presented to aid in FRAP analysis when binding varies spatially, one applying to cases of relatively fast diffusion and slow binding and the other to binding of molecules to small cellular structures. Numerical results show that using a model that represents the influential physical processes and that is formulated in the appropriate geometry can substantially improve the accuracy of FRAP calculations.     

Determination of lipid profile in meningococcal polysaccharide using reversed-phase liquid chromatography

A fast and sensitive HPLC method using fluorescence detection is developed to quantitate 1-pyrenyldiazomethane (PDAM) derivatized fatty acids derived from the lipid components of both the capsular meningococcal polysaccharide and other impurities such as endotoxin in various meningococcal vaccine samples. The HPLC method is capable of well resolving 13 relevant fatty acids within 40min by using a multi-stage acetonitrile/water gradient. Endotoxin values measured by HPLC well correlated with results from the standard Limulus amebocyte lysate (LAL) assay. Furthermore, the fatty acid profiles of various process intermediate samples as well as final purified polysaccharide products were determined to better understand and characterize the purification process.     

Mechanism of poly(ethylene glycol)-induced lipid transfer between phosphatidylcholine large unilamellar vesicles: a fluorescent probe study

Experiments were performed to assess three possible mechanisms of poly(ethylene glycol) (PEG) induced rapid lipid transfer between large unilamellar vesicles composed of dioleoylphosphatidylcholine: (1) transfer between aggregated vesicles, (2) transfer through an aqueous medium of lowered dielectric constant, and (3) transfer via a PEG carrier. The results showed that close contact between vesicles as a result of PEG dehydration was largely responsible for the rapid lipid transfer observed in the presence of PEG. The rate and extent of lipid transfer were also examined at 10 wt % PEG and analyzed in terms of a two-state model especially developed to account for the initial rate of lipid transfer as followed by the fluorescence lifetime of DPHpPC as a fluorescent lipid probe. Analysis revealed that two rate processes were involved in DPHpPC transfer between bilayers, both in the absence and presence of PEG. Since the maximum extent of transfer was 50%, transbilayer diffusion of DPHpPC seemed not to contribute to either process. The fast process in the presence of PEG was identified as due to rapid interbilayer monomer diffusion between closely apposed vesicles, and, in the absence of PEG, as due to monomer diffusion through the aqueous phase. The origin of the slow process, in both cases, remains obscure. The Arrhenius activation energies (and entropies) for the initial rates at temperatures from 10 to 48 degrees C were 15.3 +/- 0.3 kcal/mol (-26.3 +/- 0.2 eu) and 10.6 +/- 0.5 kcal/mol (-16.1 +/- 0.3 eu) in the absence and presence of PEG, respectively. The slow process was invariant with temperature.(ABSTRACT TRUNCATED AT 250 WORDS)