Static Light-scattering Measurements Research Articles

Gas−Solid Coexistence in Highly Charged Colloidal Suspensions

Aqueous suspensions of highly charged polystyrene particles with different volume fractions have been investigated for structural ordering and phase behavior using static light scattering (SLS) and confocal laser scanning microscope (CLSM). Under deionized conditions, suspensions of high-charge-density colloidal particles remained disordered whereas suspensions of relatively low charge density showed crystallization by exhibiting iridescence for the visible light. Though for the unaided eye crystallized suspensions appeared homogeneous, SLS measurements and CLSM observations have revealed their inhomogeneous nature in the form of the coexistence of voids with dense ordered regions. CLSM investigations on disordered suspensions showed their inhomogeneous nature in the form coexistence of voids with dense disordered (amorphous) regions. Our studies on highly charged colloids confirm the occurrence of gas-solid transition and are in accordance with predictions of Monte Carlo simulations using a pair-potential having a long-range attractive term [Mohanty, P. S.; Tata, B. V. R. J. Colloid Interface Sci. 2003, 264, 101]. On the basis of our experimental and simulation results, we argue that the reported reentrant disordered state [Yamanaka et al. Phys. Rev. Lett. 1998, 80, 5806 and Toyotama et al. Langmuir 2003, 19, 3236] in charged colloids observed at high charge densities is a gas-solid coexistence state.

Evidence for a Mechanism of Amyloid Formation Involving Molecular Reorganisation within Native-like Precursor Aggregates

The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive beta-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3-5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and beta-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.

Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements

The interactions of partially unfolded proteins provide insight into protein folding and protein aggregation. In this work, we studied partially unfolded hen egg lysozyme interactions in solutions containing up to 7 M guanidinium chloride (GdnHCl). The osmotic second virial coefficient (B(22)) of lysozyme was measured using static light scattering in GdnHCl aqueous solutions at 20 degrees C and pH 4.5. B(22) is positive in all solutions, indicating repulsive protein-protein interactions. At low GdnHCl concentrations, B(22) decreases with rising ionic strength: in the absence of GdnHCl, B(22) is 1.1 x 10(-3) mLmol/g(2), decreasing to 3.0 x 10(-5) mLmol/g(2) in the presence of 1 M GdnHCl. Lysozyme unfolds in solutions at GdnHCl concentrations higher than 3 M. Under such conditions, B(22) increases with ionic strength, reaching 8.0 x 10(-4) mLmol/g(2) at 6.5 M GdnHCl. Protein-protein hydrodynamic interactions were evaluated from concentration-dependent diffusivity measurements, obtained from dynamic light scattering. At moderate GdnHCl concentrations, lysozyme interparticle interactions are least repulsive and hydrodynamic interactions are least attractive. The lysozyme hydrodynamic radius was calculated from infinite-dilution diffusivity and did not change significantly during protein unfolding. Our results contribute toward better understanding of protein interactions of partially unfolded states in the presence of a denaturant; they may be helpful for the design of protein refolding processes that avoid protein aggregation.

Physicochemical properties of anionic triple-chain surfactants in alkaline solutions.

Physicochemical properties of two types of anionic triple-chain surfactants of disodium 2,10-didecyl-6-dodecyl-3,6,9-triazaundecanedioate (3C12dtda) with three hydrocarbon chains and two carboxylate headgroups, and trisodium tris(N-2-ethylamino-N'-2-dodecylcarboxylato)azane (3C12tata) with three hydrocarbon chains and three carboxylate headgroups were characterized by several techniques such as static surface tension, fluorescence spectroscopy, and dynamic and static light-scattering in alkaline solutions at pH 13. 3C12dtda shows a much lower critical micelle concentration (cmc) and higher efficiency in lowering the surface tension than 3C12tata. 3C12dtda also provides a very small occupied area per molecule as compared to 3C12tata and packs closely at the interface. The fluorescent intensity ratio of the first to third band in the emission spectra of pyrene for two triple-chain surfactants starts to decrease gradually from around the cmc obtained by the surface tension method, and decreases sharply from the concentration of 10-18 fold cmc. It is also found that the polarity of the triple-chain surfactant micelle is very much lower than that of the single-chain surfactant micelle. The results of dynamic and static light-scattering measurements show the difference behavior in the aggregation process for two triple-chain surfactants; that is, 3C12dtda forms large aggregates, and 3C12tata forms two aggregates of normal micelle size and a larger one in solution. This indicates that the aggregation states of triple-chain surfactants differ by the number of carboxylate groups, and the large aggregates are derived from the strong attractive interaction between multiple hydrocarbon chains.

Synthesis and polymerization of tail-type cationic polymerizable surfactants and hydrophobic counter-anion induced association of polyelectrolytes

A series of tail-type cationic surface-active monomers with the cationic charge at the ω-end (1; ST-C m -AB, m=5, 7, and 9, where ST is a styrenic group, C m the alkylene chain at the 4-position of styrene, and AB is alkyltrimethylammonium bromide) have been synthesized as a novel cationic polymerizable surfactant. Their fundamental physicochemical properties such as critical micelle concentration (cmc) and weight-average aggregation number of the micelle (N w(agg)) have been characterized in water at 25 °C by static light-scattering measurements. The cmc values determined for the tail-type surfactant monomers are two-orders of magnitude smaller than those of the corresponding head-type cationic surfactant monomers (2; ST-C1-AC-C m ). The N w(agg) of ST-C m -AB is 68 for m=5, 156 for m=7, and 413 for m=9. Free-radical homopolymerization of ST-C7-AB proceeds very rapidly in water as a result of organization in the micelle to afford the corresponding amphiphilic cationic polyelectrolyte with M w=3.63×106 and 23 nm hydrodynamic radius at 25 °C. Emulsion copolymerization of styrene with ST-C m -AB also proceeds rapidly to afford very stable cationic polystyrene latex particles of 30–60 nm diameter. The amphiphilic cationic polyelectrolyte of poly(ST-C7-AB) is likely to assume a compact conformation with high segment density in 0.1 mol L−1 NaCl in water. Addition of hydrophobic aromatic counter-anions with an weak acid group, for example potassium hydrogen phthalate (PHK) and sodium salicylate, to a salt-free aqueous solution of poly(ST-C7-AB) induces intermolecular aggregation and increases the solution viscosity substantially, often producing gels and precipitation at high polymer concentration.

Effect of alcohols on aqueous lysozyme–lysozyme interactions from static light-scattering measurements

Alcohols have been widely used as protein denaturants, precipitants and crystallization reagents. We have studied the effect of alcohols on aqueous hen-egg lysozyme self-interactions by measuring the osmotic second virial coefficient ( B 22) using static light scattering. Addition of alcohols increases B 22, indicating stronger protein–protein repulsion or weaker attraction. For the monohydric alcohols used in this study (methanol, ethanol, 1-propanol, n-butanol, iso-butanol and trifluoroethanol), B 22 for lysozyme reaches a common plateau at approximately 5% (v/v) alcohol, while glycerol increases B 22 more than monohydric alcohols. For a 0.05 M NaCl hen-egg lysozyme solution at pH 7, B 22 increases from 2.4×10 −4 to 4.7×10 −4 ml mol/g 2 upon addition of monohydric alcohols and to 5.8×10 −4 ml mol/g 2 upon addition of glycerol. We describe the alcohol effect using a simple model that supplements the DLVO theory with an additional alcohol-dependent term representing orientation-averaged hydrophobic interactions. In this model, the increased lysozyme repulsive forces in the presence of monohydric alcohols are interpreted in terms of adsorption of alcohol molecules on hydrophobic sites on the protein surface. This adsorption reduces attractive hydrophobic protein–protein interactions. A thicker lysozyme hydration layer in aqueous glycerol solution can explain the glycerol-increased lysozyme–lysozyme repulsion.

Physicochemical properties of quaternary ammonium bromide-type trimeric surfactants

Trimeric surfactants of quaternary ammonium bromide ( m-2-m-2-m, where m is the hydrocarbon chain length of 8, 10, or 12) with three hydrocarbon chains and three hydrophilic groups connected by two ethylene spacer chains were synthesized by the reaction of N, N, N′, N″, N″-pentamethyldiethylenetriamine and the corresponding alkyl bromide. Their physicochemical properties were characterized by surface tension, static and dynamic light-scattering, and fluorescence spectrum of pyrene techniques. The critical micelle concentrations (cmc's) of m-2-m-2-m shifted to lower concentrations with increasing hydrocarbon chain length, and their values were smaller by about one to three orders of magnitude than those of the corresponding dimeric ( m-2-m) and monomeric surfactants (C m TAB) with the same hydrocarbon chain length. Of these surfactants, 10-2-10-2-10 showed the greatest efficiency in lowering the surface tension and provided the smallest occupied area per molecule, indicating that it adsorbs more compactly at the air/water interface. In addition, from the static and dynamic light-scattering measurements, the aggregation numbers of the trimeric surfactants at the cmc were very small, and two hydrodynamic diameters above the cmc were observed.

A reversible tube-to-rod transition in a block copolymer micelle.

A remarkable morphology transition occurs with a change in temperature for a diblock copolymer [poly(ferrocenyldimethylsilane-b-dimethylsiloxane) (PFS40-b-PDMS480, PDI = 1.01)] in n-decane solution. This polymer, which forms nanotubes at 25 degrees C, rearranges to form short dense rods when the solution is heated to 50 degrees C. When the solution is cooled to 25 degrees C, the system evolves back to nanotubes. These experiments demonstrate that both structures are dynamic and represent equilibrium states of the material. Contrast matching static light-scattering measurements on the short dense rods show that the insoluble PFS core is rigid and has a length distribution similar to that seen in electron microscopy images.

Weak Gel Behaviour of Poly(vinyl alcohol)-Borax Aqueous Solutions

Thermal transition of PVA-borax aqueous gels with a PVA concentration of 60 g/L and a borax concentration of 0.28 M was investigated at temperatures ranging from 15 to 60 ○C using static light scattering (SLS), dynamic light scattering (DLS), and dynamic viscoelasticity measurements. Three relaxation modes, i.e. two fast and one slow relaxation modes, were observed from DLS measurements. Two fast relaxation modes located around 10−3∼101 sec, with one fast mode (τf1) being scattering vector q-dependent and the other fast mode (τf2, with τf2>τf1) being q-independent. The τf1 mode was attributed to the gel mode whilst the τf2 mode could be due to the hydrodynamics of intra-molecular hydrophobic domains formed by uncharged segments of polymer backbones. The slow relaxation mode with relaxation time located around 101∼103 sec in DLS data was due to the motion of aggregated clusters and was observed only at temperatures above 40 ○C. The amplitude and relaxation time of slow mode decrease as temperature is increased from 40 to 60 ○C. At temperatures below 40 ○C, no slow relaxation mode was observed. The SLS measurements showed PVA-borax-water system had fractal dimensions Df∼2.4 and Df≤2.0 as temperature was below and above 40 ○C, respectively. The simple tilting test indicated gel behaviour for the PVA-borax aqueous system at temperatures below 40 ○C with a creep flow after a long time exposure in the gravity field. But the dynamic viscoelasticity measurements demonstrated a solution behaviour for PVA/borax/water at temperatures below 40 ○C, the critical gel point behaviour for G′(ω) and G″(ω) was not observed in this system as those reported for chemical crosslinked gels. These results suggest that the PVA-borax aqueous system is a thermoreversible weak gel.

Structural and dynamic properties of colloidal liquids and gases of silica spheres (29 nm in diameter) as studied by the light scattering measurements

Static and dynamic light-scattering measurements are made for colloidal-liquids and -gases of silica spheres (29 nm in diameter) in the exhaustively deionized aqueous suspension and in the presence of sodium chloride. Single broad peak is observed in the light-scattering curve and the liquid-like and gas-like distributions have been observed. Colloidal crystals are not formed at any sphere concentrations. The nearest-neighbored interparticle distances of colloidal liquids, l obs , agree excellently with the effective diameters of spheres (d eff ) including the electrical double layers in the effective soft-sphere model and also with the mean intersphere distances, l o , calculated from the sphere concentration, i.e., l obs ≈d eff ≈ l o . This relation supports the importance of the electrostatic interparticle repulsive interaction. Two dynamic processes have been extracted separately from the time profiles of autocorrelation function of colloidal liquids. Decay curves of colloidal gases are characterized by the single translational diffusion coefficients, which are always lower than the calculation from the Stokes-Einstein equation using true diameter of spheres and increase as ionic concentration increases. These experimental results emphasize the importance of the expanded electrical double layers and the electrostatic intersphere repulsion on the structural and dynamic properties of the colloidal liquids and gases.

Light scattering and crossover critical phenomena in polymer solutions

An accurate photon-correlation spectrometer capable of measuring scattered light at two fixed scattering angles simultaneously in the temperature range from 10 to 120 degrees C is described. For the detection of the correlation function of the scattered light both an original correlator (PhotoCor-SP) with a linear-time-scale channel spacing and an ALV GmbH correlator (Model ALV-5000) with a logarithmic-time-scale channel spacing are used. High-resolution static and dynamic light-scattering measurements near the critical point of a polystyrene solution were performed with this instrument. The static-intensity measurements reveal that the crossover from mean-field to Ising behavior occurs at a temperature at which the correlation length of the critical fluctuations becomes equal to the radius of gyration of the polymer molecule. We found that the wave-number dependence and the temperature dependence of the diffusion coefficient of the concentration fluctuations are not consistent with existing theory for critical dynamics in low-molecular-weight liquid mixtures.

Genetic Algorithm Approach to the Determination of Particle Size Distributions from Static Light-Scattering Data

The application of genetic algorithms to the inversion of static light-scattering (SLS) measurements is investigated. The approach is illustrated using simulated data on samples with unimodal, bimodal, and trimodal distributions of spherical particles. Particles having diameters in the range from 100 to 4000 nm are treated. Results are compared with other inversion procedures. It is found that genetic algorithms yield very good results in this situation.

Kinetics of the Coil−Globule Transition of Poly(methyl methacrylate) in a Mixed Solvent

The coil−globule transition and chain collapse process were studied for poly(methyl methacrylate) in the mixed solvent tert-butyl alcohol + water (2.5 vol %) by static light-scattering measurements. The expansion factor α2 for the mean-square radius of gyration was obtained as a function of time for various molecular weights. For the molecular weights Mw × 10-6 = 1.57, PMMA chains collapsed to an equilibrium globule within 30 min after quench. For Mw × 10-6 = 4.0 and 12.2, chain collapse processes were observed for time periods of hours and days, respectively. The time dependence of the observed expansion factor was represented by the stretched exponential form α2 = α2∞ + (1 − α2∞) exp[−(t/τ*)β], where α2∞ is the equilibrium expansion factor sufficiently long time after quench. β was obtained in the range from 0.1 to 0.4 and decreased with decreasing temperature and increasing molecular weight. From the behavior of β, the stretched exponential process was suggested to be intrinsic to each polymer chain rather than due to a superposition of exponentially collapse processes of polymer chains with various decay times. The equilibrium expansion factor obtained as a function of temperature and molecular weight was represented by the theoretical prediction α3 − α − C(α -3 − 1) = B(1 − θ/T)M1/2. The constants B and C were determined to be B = 0.0164 and C = 0.049 independent of the molecular weight.

Chain Aggregation and Chain Collapse of Poly(methyl methacrylate) in a Mixed Solvent

Chain aggregation processes were studied for dilute solutions of PMMA with the molecular weight m = 1.57 x 10 6 g/mol in the mixed solvent tert-butyl alcohol + water (2.5 vol %) at 25.0 and 30.0 °C in the concentration range from 1.4 x 10 -4 to 5.7 x 10 -4 g/cm 3 . The weight-average molecular weight w and z-average squared radius 2 of clusters of polymer chains were determined as a function of time by static light-scattering measurements. The phase separation temperature increased from 35.7 to 36.7 °C in the concentration range. The aggregation process near the concentration c = 1.46 x 10 -4 g/cm 3 was observed for a time period of about 10 000 min at 25.0 °C and 2000 min at 30.0 °C. At each temperature both the plots of In w and In 2 vs t (min) were represented by curved lines. The initial slopes of the lines were proportional to the concentration c. Accordingly, the scaled relations of w /M(0) = exp(gct) and z /R 2 (0) = exp(hct) were obtained at small ct with g = 5.01 cm 3 /(g min) and h = 3.97 cm 3 /(g min) at 25.0 °C and with g = 16.9 and h = 13.2 at 30.0 °C, where M(0) and R 2 (0) are the values extrapolated to t = 0. The reciprocals of gc and hc give the characteristic times of the chain aggregation. The longer characteristic times at 25.0 °C than at 30.0 were attributed to the effect of the chain collapse. The exponential growth indicated a reaction-limited cluster aggregation of polymer chains. The double-logarithmic plot of w vs z 1/2 at each temperature was independent of concentration and represented by a single straight line with the slope D = 2.53 ± 0.02 at 25.0 °C and 2.66 ± 0.01 at 30.0 °C.

Effects of PEG on detergent micelles: implications for the crystallization of integral membrane proteins.

The solubilization of integral membrane proteins with detergents produces protein-detergent complexes (PDCs). Interactions between the detergent moieties of PDCs contribute significantly to their behavior. The effects of the precipitating agent polyethylene glycol (PEG) upon these detergent-detergent interactions have been examined, focusing on the detergent system used to crystallize the bacterial outer membrane protein OmpF porin. Static and dynamic light scattering were used to assess the effects of temperature and concentration upon the hydrodynamic size distribution and the aggregation state of detergent micelles and a phase diagram for micellar solutions was mapped. Estimates of the second osmotic virial coefficient obtained from static light-scattering measurements on micelles were shown to accurately reflect the thermodynamic quality of the solvent. Solvent quality decreases as the consolute boundary is approached, suggesting micelle-micelle attractive forces help to organize PDCs into crystalline aggregates near the cloud point. An apparent increase in micelle mass is observed as the solution approaches the cloud point. These results raise the possibility that the detergent-mediated aggregation of PDCs and/or slight changes in micelle geometry may prove to be important in the nucleation of membrane protein crystals.

Physicochemical consequences of the perdeuteriation of glutathione S-transferase from S. japonicum.

Glutathione S:-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9 degrees C, whereas that for dGST was 51.0 degrees C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K(m) to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.

Chain aggregation in dilute solutions of poly(methyl methacrylate) below the phase-separation temperature.

For dilute solutions of poly(methyl methacrylate) in isoamyl acetate with the molecular weight M(w)=4.4 x 10(6), the phase-separation process was studied by static light-scattering measurements. The dilute solutions in the concentration range from 1.4 x 10(-4) to 3.8 x 10(-4) g/cm(3) were quenched to about 16 K below the phase-separation temperature, and the aggregation processes of polymer chains were measured over a period of several hours. By analyzing the light-scattering data with the Guinier plot, the weight-averaged molecular weight <M>(w) and z-averaged square radius <R2>(1/2)(z) for clusters of polymer chains were determined as a function of time t(min) and concentration c (g/cm(3)). The growth of clusters was represented by the exponential forms <M>(w)/M(0)=e(gct) and <R2>(z)/R(2)(0)=e(hct) as a function of ct, where M(0) and R2(0) were the values at t=0, and the constants g and h were determined to be g=11.6 and h=7.5. A double-logarithmic plot of <M>(w) versus <R2>(1/2)(z) yielded a straight line with the slope D=3.06+/-0.02. These characteristic features of the chain aggregation process were compared with the Smoluchowski equation for cluster-cluster aggregation with the collision kernel (i+j) for i-mer and j-mer. The observed slow growth of clusters was attributed to the reaction- limited cluster aggregation. The chain density in a cluster was found to increase with an increase of the cluster size, resulting in the slope D exceeding 3.

Mie light-scattering granulometer with an adaptive numerical filtering method II Experiments

A nephelometer is presented that theoretically requires no absolute calibration. This instrument is used for determining the particle-size distribution of various scattering media (aerosols, fogs, rocket exhausts, engine plumes, and the like) from angular static light-scattering measurements. An inverse procedure is used, which consists of a least-squares method and a regularization scheme based on numerical filtering. To retrieve the distribution function one matches the experimental data with theoretical patterns derived from Mie theory. The main principles of the inverse method are briefly presented, and the nephelometer is then described with the associated partial calibration procedure. Finally, the whole granulometer system (inverse method and nephelometer) is validated by comparison of measurements of scattering media with calibrated monodisperse or known size distribution functions.

Mie light-scattering granulometer with adaptive numerical filtering I Theory

A search procedure based on a least-squares method including a regularization scheme constructed from numerical filtering is presented. This method, with the addition of a nephelometer, can be used to determine the particle-size distributions of various scattering media (aerosols, fogs, rocket exhausts, motor plumes) from angular static light-scattering measurements. For retrieval of the distribution function, the experimental data are matched with theoretical patterns derived from Mie theory. The method is numerically investigated with simulated data, and the performance of the inverse procedure is evaluated. The results show that the retrieved distribution function is quite reliable, even for strong levels of noise.

A spectroscopic analysis of thermal stability of the Chromobacterium viscosum lipase

The thermal stability of the lipase from Chromobacterium viscosum was assessed by deactivation (loss of activity), fluorescence, circular dichroism (CD) and static light scattering (SLS) measurements. Lipase fluorescence emission is dominated by the tryptophyl contribution. An increase in the tyrosyl contribution from 2 to 16% was only observed upon prolonged incubation at 60°C. The effect of temperature on the tryptophyl quantum yield was studied and two activation energies were calculated. Tryptophan residues in the native structure have an activation energy of 1.9 kcal mol −1 for temperature-dependent non-radiative deactivation of the excited state. A structural change occurs at approximately 66.7°C and the activation energy increases to 10.2 kcal mol −1. This structural change is not characterized by tryptophan exposure on the surface of the protein. The deactivation and the evolution of structural changes with time after lipase incubation at 60°C were assessed by fluorescence, CD and SLS measurements. CD spectra show that both secondary and tertiary structures remain native-like after incubation at 60°C in spite of the fluorescence changes observed (red-shift from 330 to 336 nm on the trytophyl emission). SLS measurements together with the CD data show that deactivation may be due to protein association between native molecules. Deactivation and the decrease on the fraction of non-associated native lipase evaluated by changes in fluorescence intensity with time, show apparent first order kinetics. According to the rate constants, fluorescence changes precede deactivation pointing to an underestimation of the deactivation. Reactivation upon dilution during the activity assay and substrate-induced reactivation due to lipase interfacial adsorption are possible causes for this underestimation.