Surface and Aggregation Behavior of Pentablock Copolymer PNIPAM7-F127-PNIPAM7 in Aqueous Solutions.

The triblock Pluronic F127 was modified by introducing poly(N-isopropylacrylamide) (PNIPAM) at both the poly(ethylene oxide) ends, and the pentablock copolymer so-prepared was characterized by gel permeation chromatography and (1)H NMR. The degree of polymerization of NIPAM blocks at the two ends was 7. The solution behavior and microstructure of copolymer aggregates in water and aqueous salt solution were examined and compared with F127 by UV-visible absorption spectroscopy, microdifferential scanning calorimetry, dynamic light scattering (DLS), and small-angle neutron scattering (SANS). The behavior of the pentablock copolymer at the air/water interface was determined by Langmuir film balance. Two lower critical solution temperatures were observed for pentablock copolymer, corresponding to poly(propylene oxide) and PNIPAM blocks, respectively. DLS studies show that micelle size increased with increase in temperature and in the presence of salt. SANS measurements provided temperature-dependent structural evolution of copolymer micelles in water and salt solution. The copolymer displays an isotherm with four classical regions (pancake, mushroom, brush, and condensed state). The study has potential applications in controlled drug delivery due to the tunable phase behavior and biocompatibility of the copolymer.

Investigations on microstructural changes in pH responsive mixed micelles of Triton X-100 and bile salt.

Aqueous solution behaviour of a nonionic surfactant Triton X-100 is investigated in the presence of two bile salts namely sodium deoxycholate (NaDC) and sodium cholate (NaC) at different pH, temperatures and in the presence of sodium chloride and the resultant structural changes to accordingly formed mixed micelles were analyzed by using cloud point (CP), viscosity and scattering techniques. Both the bile salts increased the CP and showed a corresponding decrease in viscosity and apparent hydrodynamic diameter (Dh), which can further be subsided with the progressive addition of sodium chloride and an increase in temperature. Interestingly, in the presence of bile salt below pH ∼5, CP decreased with corresponding increase in viscosity, while a reversed trend was observed above pH∼8. Small angle neutron scattering data reveal that nearly spherical mixed micelles were formed in the presence of bile salt which grow and transform to prolate ellipsoidal ones at pH∼3. These morphological changes are facilitated by the protonation of carboxylic acid group of bile salt and deeper penetration of bile acid molecules into TX-100 micelles at lower pH. Proposed molecular interactions are extremely informative to understand more about these biologically important compounds playing a crucial role in digestion processes.

Butanol solubilization in aqueous F127 solution: investigating the enhanced micellar solvation and consequent improvement in gelation characteristics.

Pluronics(®) are an important class of non-ionic surfactants because of their rich phase behavior and numerous industrial and biomedical applications. F127, an FDA approved Pluronic(®) is the most prominent member amongst them owing to its potential uses as vehicle for drug delivery and template for the fabrication of mesoporous materials. A cubic micellar gel formed by this copolymer above 15 wt% concentration is the commonly used form of self assembled structure for these applications. In this manuscript we report SANS, fluorescence and rheological studies on the effect of n-butanol on gelation characteristics of aqueous F127 solutions. The studies show that solubilization of n-butanol results in a large increase in viscosity of micellar solution at a fixed copolymer concentration, and leads to the formation of stiff gel at F127 concentration as low as 9 wt%. SANS and fluorescence studies attribute this to enhancement in micellar solvation due to solubilization of n-butanol. Quite interestingly, SANS studies show that n-butanol induced F127 gels form at significantly lower micellar volume fraction than the pure F127 gels. The observed improvement in gelation characteristics can have important bearing with the application in making mesoporous materials since n-butanol is used as co-surfactant to control pore size of such structures formed with F127 gels as template.

Solubilization of parabens in aqueous Pluronic solutions: investigating the micellar growth and interaction as a function of paraben composition.

The influence of methyl paraben (MP) and butyl paraben (BP) on the aggregation characteristics of Pluronics in an aqueous medium has been investigated by DLS, SANS, viscometry, and fluorescence measurement techniques. Parabens are extensively used as preservatives in cosmetic, pharmaceutical, and food products. In this paper, we show that their influence on the restructuring and growth of Pluronics micelles vary quite significantly with their aqueous solubility and with the composition of Pluronics. In the case of P105 and P104, MP reduces the sphere-to-rod transition temperature down to room temperature, but BP with significantly less aqueous solubility than MP suppresses such micellar transition and leads to the formation of micellar clusters due to the onset of intermicellar attractive interaction. In the case of more hydrophobic Pluronic P103, on the other hand, both MP and BP are able to induce rapid room temperature sphere-to-rod micellar growth, which is not observed in the presence of water structure making salts like NaCl and Na(3)PO(4). These observations have been attributed to modulation of growth and restructuring processes of the Pluronic micelles arising due to different locations of parabens within the micellar corona as determined by their aqueous solubility and the hydrophobicity of the Pluronics.

Interaction between the ionic liquids 1-alkyl-3-methylimidazolium tetrafluoroborate and Pluronic® P103 in aqueous solution: a DLS, SANS and NMR study.

The effect of three ionic liquids (ILs) 1-alkyl 3-methyl imidazlolium tetraflouroborates (C(n)mim BF(4)n=4, 6, 8) on micellar solutions of an ethylene oxide-propylene oxide block copolymer (PEO-PPO-PEO), Pluronic® P103 was examined from scattering and NMR techniques. The ILs alter the cloud point and micelle size dependant on their alkyl chain length and the results are discussed in terms of their behavior as cosolvent/cosurfactant. Cloud point data support the hydrogen bonding between the imidazolium cation and P103 while dynamic light scattering (DLS) and small angle neutron scattering (SANS) reveal that presence of ionic liquid is not conducive to the micelle formation of P103. The selective nuclear Overhauser effect (NOESY) indicates that the PPO block of the P103 interacts with the alkyl group of the C(n)mim(+) cation by hydrophobic interaction. Through this kind of interactions, C(n)mim BF(4) and P103 can form mixed micelles. This result indicates that the presence of ILs hinders the micelle formation of P103 in solution and promotes P103 to orient at air/water interface.

Phenol solubilization in aqueous Pluronic® solutions: investigating the micellar growth and interaction as a function of Pluronic® composition.

Pluronics® are considered as potential materials for the removal of contaminants like phenol from polluted water sources because of their superior solubilizing capacity of aromatic compounds. Systematic studies on the influence of solubilization of phenol on room temperature aggregation characteristics of Pluronics® in water are, however, conspicuous by their absence. In this manuscript, we thus report DLS, SANS and rheological studies on the influence of phenol on the aggregation characteristics of four Pluronics® viz. F127, P123, P104 and P103. The aim of this study has been to understand the role played by the composition of the Pluronics® in determining growth and interaction of the micelles induced by solubilization of phenol. The study shows that in the case of F127 and P123, phenol solubilization leads to a large increase in light scattering intensity due to an onset of attractive intermicellar interactions and consequent formation of micellar clusters. P123 being smaller than F127 shows a subsequent time dependent micellar growth, leading to a sphere-to-rod shape transitions in micelles. The copolymers P103 and P104, which are smaller and less hydrophobic than P123, respectively, exhibit a large increase in solution viscosity in the presence of phenol owing to a rapid sphere-to-rod micellar growth. The observation of such a fine interplay between the growth and interaction of the pluronic micelles in the presence of a hydrophobic solvent is first of its kind and highlights the role of composition of pluronic in determining the kinetics of the micellar restructuring process.

Spectral and hydrodynamic studies on p-toluidine induced growth in cationic micelle.

The effect of p-toluidine (PTD) on the growth of cationic surfactant micelles in aqueous solutions was examined by viscosity, UV-visible spectroscopy, dynamic light scattering (DLS), (1)H NMR and nuclear Overhauser effect spectroscopy (NOESY). Viscosity and scattering results are used to follow the growth of the aggregates and examine the structural transitions that occur. The reduction of electrostatic repulsions between the surfactant headgroups in combination with the hydrophobicity of the additive caused micellar growth and viscoelasticity at high PTD concentration; the micellar growth was found to be more pronounced in the presence of salt. (1)H NMR chemical shift changes reflect the interaction of PTD molecules with surfactant micelles while 2D NOESY experiments reveal that PTD molecules insert themselves in micelles such that its -CH(3) group is in contact with the core region and the benzene ring resides near the palisade layer of micelle.

Growth and interaction of the Tetronic 904 micelles in aqueous alkaline solutions.

The influence of sodium hydroxide (NaOH) on the aggregation characteristics of the Tetronic 904 (T904) has been studied in the aqueous medium by dynamic light scattering (DLS), small angle neutron scattering (SANS), and viscometry methods. This polyethylene oxide (PEO)-polypropylene oxide (PPO) based X-shaped triblock copolymer shows pH sensitive aggregation characteristics due to the presence of a central amine group attached with the PPO block. The aqueous micellar solutions of this copolymer have been found to show a large increase in the room temperature (30 °C) relative viscosity with an increase in NaOH concentration, before they undergo phase separation at 1.25 M NaOH concentration. SANS and DLS studies ascribe this behavior to a sphere to rod growth of the copolymer micelles on approaching the cloud points of the copolymer solutions. DLS studies show that the observed micellar growth is accompanied by critical scattering due to the onset of an attractive intermicellar interaction. To understand the role of NaOH in inducing the observed micellar shape transition, we have also studied the effect of NaOH on the micellar solutions of Pluronic P84, which does not show pH sensitivity due to the absence of the central amine group but has the same weight fraction of the PEO block (40%) as that of T904. Quite interestingly, the P84 micelles too have been found to undergo a similar room temperature sphere to rod shape transition in the presence of NaOH. The observed growth and interaction of the Tetronic micelles have thus been attributed to the dual effects of NaOH in influencing the aggregation characteristics of the Tetronic molecules. On one hand, it stabilizes these micelles by the deprotonation of the copolymer molecules, and on the other hand, its dehydrating effect on the copolymer molecules promotes the onset of micellar growth and intermicellar attractive interaction at the room temperature. The observed results are the first of their kind in the aqueous Tetronic system.

Micellization and solubilization of a model hydrophobic drug nimesulide in aqueous salt solutions of Tetronic T904.

The micellar and phase behavior of an ethylene oxide-propylene oxide branched octablock copolymer Tetronic T 904 (hereafter written as T904) in water and NaCl solutions was examined. The copolymer shows a cloud point (CP) ranging from 74-65°C in the concentration range of 1-10% and forms aggregates (micelles) with a hydrodynamic diameter around 10-12nm in the temperature range 30-40°C. Stable, bluish solutions containing aggregates of variable size (several hundred nm in some cases) were observed even at temperatures much less than the critical micellization temperature (CMT=30°C for a 2% solution in water). The CP and the CMT markedly decrease in the presence of NaCl due to the dehydration of the polyethylene oxide shell. The size of the micelles in water or salt solutions increases at temperatures close to the CP as inferred from viscosity measurments. A model drug compound (nimesulide, NIM) was solubilized in T904 micelles which showed a remarkable increase in solubilization at higher temperature; however, a decrease in solubilization was observed in salt solutions. The thermodynamic parameters for solubilization were obtained, and the location of NIM in the copolymer micelles was investigated by UV-Visible spectroscopy.

Induced micellization and micellar transitions in aqueous solutions of non-linear block copolymer Tetronic® T904.

Tetronics® (Poloxamines) are the least studied block copolymers with an X-shaped molecular geometry formed by four poly (propylene oxide, PPO) and poly (ethylene oxide, PEO) block chains, bonded to a central ethylenediamine group. Compared to their linear counterparts, the Pluronics, Tetronics® are novel, in that they possess superior physicochemical properties, and are relatively less studied. A complete understanding of their solution behavior under different solution conditions can make them competitive candidates for novel drug delivery systems. The micellization behavior and aqueous solution properties of Tetronic® T904 [(EO(15)PO(17)) (2)NCH(2)CH(2)N (PO(17)EO(15))(2)] have been determined by cloud point, viscosity, dynamic light scattering (DLS), small-angle neutron scattering (SANS), and nuclear magnetic resonance (NMR) measurements. The copolymer formed spherical micelles at 30°C with a core radius (R(c)) of about 2.5 nm, a hard sphere radius (R(hs)) of 5.2 nm and an aggregation number (N(agg)) of 10. The effect of copolymer concentration, temperature, pH and salt on the micellar and phase behavior is examined. The copolymer has been found to exist in aggregated form only at higher pH values of >8. An increase in micelle size and aggregation has been observed for an increase in temperature and salt concentration, mainly due to the dehydration of the hydrated PEO shell. The added salts (NaCl, Na(2)SO(4) and Na(3)PO(4)) induce micellization and favor the micellar transition at lower temperatures due to the "salting out effect"; the effect of anions follows the Hofmeister series (PO(4)(3-)>SO(4)(2-)>Cl(-)).

Time dependent sphere-to-rod growth of the pluronic micelles: investigating the role of core and corona solvation in determining the micellar growth rate.

The salt induced sphere-to-rod growth in the micelles of the PEO-PPO triblock copolymers, Pluronic P123 (EO(20)PO(70)PEO(20)) and Pluronic P103 (EO(16)PO(61)PEO(16)), has been studied by dynamic light scattering (DLS), viscometry, and small angle neutron scattering (SANS) techniques. The observed micellar growths are found to be time dependent and have a strong variation in their growth rate with changing anion type and copolymer composition. The rate of growth increases rather significantly with an increase in the water structure making abilities of the anions along the Hofmeister series in the order Cl(-) < F(-)< (PO(4))(3-). This has been attributed to an increasing ability of these ions to dehydrate the micellar corona, a factor that plays an important role in inducing sphere-to-rod shape transition of the micelles. The copolymer composition also has a significant influence on the micellar growth rate, as the P103 with a smaller molecular weight than P123 shows a significantly faster growth of its micelles under similar conditions. The observed time dependence in micellar growth in these systems has been attributed to a slow micellar restructuring process necessary to attain the equilibrium structure of the micelles. A remarkable improvement in the growth rate of the micelles, however, could be achieved in the presence of ethanol, a solvent that has affinity toward both the PPO and PEO blocks. Our spectroscopic studies suggest that the observed improvement in the micellar growth rate by ethanol is due to an accelerated restructuring process of the micelles in the presence of the solvated micellar core. These studies thus highlight the role of changing core and corona solvation characteristics of the pluronic micelles in determining their rearrangement and the growth rate, which is first of its kind in the aqueous pluronic system.

Viscoelastic micellar water/CTAB/NaNO(3) solutions: rheology, SANS and cryo-TEM analysis.

Aqueous micellar solutions of the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) and sodium nitrate (NaNO(3)) were examined using steady and dynamic rheology, small-angle neutron scattering (SANS) and cryogenic-transmission electron microscopy (cryo-TEM). Upon addition of NaNO(3), the CTAB spherical micelles transform into long, flexible wormlike micelles, conveying viscoelastic properties to the solutions. The zero-shear viscosity (eta(0)) versus NaNO(3) concentration curve exhibits a well-defined maximum. Likewise, upon increase in temperature, the viscosity decreases. Dynamic rheological data of the entangled micellar solutions can be well described by the Maxwell model. Changes in the structural parameters of the micelles with addition of NaNO(3) were inferred from SANS measurements. The intensity of scattered neutrons at the low q region was found to increase with increasing NaNO(3) concentration. This suggests an increase in size of the micelles and/or decrease of intermicellar interactions with increasing salt concentration. Analysis of the SANS data using prolate ellipsoidal structure and Yukawa form of interaction potential between micelles indicates that addition of NaNO(3) leads to a decrease in the surface charge of the ellipsoidal micelles and consequently an increase in their length. The structural transition from spherical to entangled threadlike micelles, induced by the addition of NaNO(3) to CTAB micelles is further confirmed by cryo-TEM.

Influence of a hydrophobic diol on the micellar transitions of Pluronic P85 in aqueous solution.

Micellization behavior of an amphiphilic ethylene oxide-propylene oxide-ethylene oxide tri-block copolymer Pluronic P85 [(EO)(26)(PO)(39)-(EO)(26)] in aqueous solution and in the presence of a hydrophobic C(14)diol (also known as Surfynol104) was examined by physico-chemical methods such as viscometry, cloud point (CP) and scattering techniques viz. dynamic light scattering (DLS) and small angle neutron scattering (SANS). The addition of diol decreases the cloud point and gelation temperature of aqueous Pluronic P85 copolymer solution. DLS and SANS measurements of the polymer in aqueous solution indicated micellar growth and sphere to rod transition in the presence of diol. Surfynol 104 is a sparingly water soluble diol surfactant with a solubility of approximately 0.1 wt%. However, up on addition to Pluronic solution, diol gets incorporated in the block copolymer micelles and leads to structural transition of the micelles. An increase in the temperature and the presence of added sodium chloride in the solution further enhances this effect. The addition of hydrophobic C(14)diol increases the hydrodynamic size and aggregation numbers of the micellar system. The micellar parameters for the copolymer in the presence of C(14)diol are reported at different temperatures and added sodium chloride concentrations.

Effect of phenol on the aggregation characteristics of an ethylene oxide-propylene oxide triblock copolymer P65 in aqueous solution.

The effects of phenol on the micellization, micellar growth, and phase separation of a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-PPO-PEO) amphiphilic copolymer (Pluronic P65: EO19 PO30 EO19) in aqueous solution have been studied by cloud point, viscosity, dynamic light scattering (DLS), differential scanning calorimetry (DSC), fluorescence spectroscopy, and small-angle neutron scattering (SANS). Various concentrations of P65 have been chosen to estimate the effect of phenol on different concentration regions of P65. Phenol interacts quite differently at low concentrations (0-2%) than at high concentrations (2-10%) of P65, as per the observation that phenol is more predominant at smaller concentrations of P65. A marked decrease in the cloud points of the P65 solutions is observed in presence of phenol. The critical micelle temperature (CMT) of P65 shows a synergistic effect of phenol on P65 aggregates. Micellar transitions, phase separation, and aggregation behaviours like micellization and micellar growth in the presence of phenol have been observed by combining viscometry, DLS, DSC, and CP. DLS shows that the effect of phenol is predominant at high temperatures. SANS shows a high increase in axial ratio and aggregation numbers in the presence of phenol at fixed concentrations of P65. Fluorescence data illustrate that addition of phenol makes micelles polar but at the same time its favours aggregation. Water-soluble phenol (present in low concentrations) forms aggregates with P65, which can be separated by cloud point extraction, making this study interesting for separation of phenol from the phenol-water system.

Interaction of urea with pluronic block copolymers by 1H NMR spectroscopy.

Solution 1H NMR techniques were used to characterize the interaction of urea with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers. The urea was established to interact selectively with the PEO blocks of the block copolymer, and the interaction sites were found not to change with increasing temperature. Such interactions influence the self-assembly properties of the block copolymer in solution by increasing the hydration of the block copolymers and stabilizing the gauche conformation of the PPO chain. Therefore, urea increases the critical micellization temperature (CMT) values of PEO-PPO-PEO copolymers, and the effect of urea on the CMT is more pronounced for copolymers with higher PEO contents and lower for those with increased contents of PPO segments.

A phenol-induced structural transition in aqueous cetyltrimethylammonium bromide solution.

The effect of phenol on the structure of micellar solution of a cationic surfactant, cetyltrimethylammonium bromide (CTAB) was investigated using viscosity, dynamic light scattering (DLS), small angle neutron scattering (SANS) and nuclear magnetic resonance (NMR) techniques. The relative viscosity and apparent hydrodynamic diameters of the micelles in CTAB solution increase initially and then decrease with addition of phenol. SANS studies indicate a prolate ellipsoidal structure of the micelles. The axial ratio of the prolate ellipsoidal micelles increases and then decreases with addition of phenol, consistently with DLS and viscosity measurements. NMR studies confirm the solubilization of phenol to the palisade layer and growth of the micelles at high concentration of phenol as revealed from the broadening of peaks.

Interaction of cationic surfactants with carboxymethylcellulose in aqueous media.

We have examined the polymer-surfactant interaction in mixed solutions of the cationic surfactants, i.e., dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, tetradecyltriphenylphosphonium bromide, and tetradecylpyridinium bromide and a semiflexible anionic polyelectrolyte carboxymethylcellulose in water and aqueous salt solutions by various techniques: tensiometry, viscosimetry or ion-selective electrode method, and dynamic light scattering. We have investigated the effect of varying surfactant chain length, head group size, counterion, and ionic strength on the critical aggregation concentration (CAC) of mixed polymer surfactant systems and the collapse of the polymer molecule under different solution conditions. The CAC decreases with increasing alkyl chain length. Above a certain surfactant concentration, mixed aggregates start growing until their macroscopic phase separation. The growth is more rapid with greater surfactant tail length and with increasing head group size. This is attributed in both cases to the increasing hydrophobic interaction between polymer and surfactant. Among surfactants with monovalent halide counterions, iodide induces the strongest binding, reflected by the onset of growth of the mixed aggregates at low surfactant concentration. This is perhaps related to the decreasing hydration of the counterion from chloride to iodide. The surfactant concentration at which the viscosity of the solution starts to decrease sharply is smaller than the CAC, and probably reflects polymer chain shrinkage due to noncooperative binding.

Concentration, temperature, and salt-induced micellization of a triblock copolymer Pluronic L64 in aqueous media.

The effect of copolymer concentration, temperature, and sodium halides (NaI, NaBr, NaCl, and NaF) on micellization and micellar properties of a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-PPO-PEO) amphiphilic copolymer (Pluronic L64: EO13PO30EO13), was examined by different methods such as dye spectral change, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), small angle neutron scattering (SANS), dynamic light scattering (DLS), viscosity, and cloud point (CP). Temperature/polymer concentration/salt dependent aggregation behavior of L64 was observed. The data on critical micelle concentration (CMC), critical micelle temperature (CMT), (CP), micelle size, and shape are reported. The Fourier transform infrared (FTIR) showed temperature dependent changes in C-O-C stretching variation band towards higher wave numbers and broadening of band width during the micellization process; this was attributed to increase in proportion of the anhydrous methyl groups, while the proportion of the hydrated methyl groups was decreased. Differential scanning calorimetry (DSC) provides CMTs and CPs from the same experiment. CMC values derived from dye spectral change, decrease significantly with the addition of salt. The increases in salt/copolymer concentration lower the onset temperature of micellization (CMT). Halide anions influence both CMT and CP in the order of F- > Cl- > Br- > I- when total salt and copolymer concentration kept constant. SANS results show the increase of inter-micellar interaction due to the increase in temperature/salt concentration; this is supported by viscosity data.

Cationic surfactant/bile salt interaction studied by fluorescence quenching.

Fluorescence quenching measurements were performed on aqueous solutions of the cationic surfactant cetyltrimethylammonium halide (CTAX) and two bile salts, sodium cholate (NaC) and sodium deoxycholate (NaDC), to study the state of aggregation in the mixtures. Pyrene was used as a photoluminiscence probe in the study, and dimethylbenzophenone (DMBP) as the quencher. Analysis of time-resolved decay data with and without quencher using a simple kinetic model gave information of the different aggregation characteristics in the above two cases. Mixed micelles of CTAX/NaC were small and spherical at all compositions, while those of CTAX/NaDC tended to grow from spherical micelles to larger rod-like mixed aggregates at equimolar and close-to-equimolar concentrations. In the latter case more complex kinetics ensues and the fluorescence decays were treated using a generatized model for diffusion-controlled quenching along one dimension for infinitely long rod-like micelles. The mutual diffusion coefficient for the probe-quencherpair was determined.

Stability and kinetic behaviour of some enzymes in surfactant environment.

The interaction of alpha-chymotrypsin, invertase, alcohol dehydrogenase and alkaline phosphatase with some ionic and non-ionic surfactants, viz. sodium dodecyl sulphate, dioctyl sodium sulphosuccinate, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide and Triton X-100, has been examined by studying the effect of varying surfactant concentrations on enzyme activities as well as by determining the time-dependent inactivation and the time-independent inhibition. The kinetic parameters, Km and Vmax, for alpha-chymotrypsin-catalysed reaction in presence of sodium dodecyl sulphate were evaluated. Anionic surfactants markedly decreased enzyme activity, whereas cationic surfactants were less effective. Nonionics showed no effect. This change in enzyme activity was also dependent on the nature of enzyme.