A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal

Equilibrium and kinetic aspects of Triton X-100 adsorption onto hydrophobic Bio-Beads SM2 were investigated in detail using the batch procedure originally described by Holloway, P.W. (1973) Anal. Biochem. 53, 304-308. The results demonstrated the importance of the initial detergent concentration, the amount of beads, the commercial source of the detergent, the temperature and the presence of phospholipids in determining the rates of Triton X-100 adsorption onto Bio-Beads. One of the main findings was that Bio-Beads allowed the almost complete removal of Triton X-100, whatever the initial experimental conditions. It was shown that monomeric as well as micellar detergent could be adsorbed and that a key factor in determining the rate of detergent removal was the availability of the free bead surface. Rates of detergent removal were found to be linearly related to the amount of beads even for bead concentrations above those sufficient to remove all the detergent initially present. Adsorptive capacity of phospholipids onto Bio-Beads SM2 was also analyzed and found to be much smaller (2 mg lipid per g of wet beads) than that of Triton X-100 (185 mg TX 100 per g of wet beads). A more general aspect of this work was that the use of Bio-Beads SM2 provided a convenient way for varying and controlling the time course of Triton X-100 removal. The method was further extended to the formation of liposomes from phospholipid-Triton X-100 micelles and the size of the liposomes was found to be critically dependent upon the rate of detergent removal. A general procedure was described to prepare homogeneous populations of vesicles. Freeze-fracture electron microscopy and permeability studies indicated that the liposomes thus obtained were unilamellar, relatively large and impermeable. Noteworthy, this new procedure was shown to be well suited for the reconstitution of different membrane transport proteins such as bacteriorhodopsin, Ca2(+)-ATPase and H(+)-ATPase.     

Stabilization of fibroblast growth factors by a non-cytotoxic zwitterionic detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (chaps)

Summary The potential usefulness of a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), in the stabilization of acidic and basic fibroblast growth factors (FGFs) was examined. Among several detergents, CHAPS was found to be not only non-cytotoxic but also most useful in handing the diluted preparations of FGFs. The advantages are as follows: 1) at lower concentrations than 0.01% CHAPS did not affect growth factor activity of calf serum (CS) and the growth rate of BLAB/c 3T3 cells. The primary culture of rat prostate epithelium and colony formation of NRK-49F cells were hardly influenced by CHAPS lower than 0.003%; 2) the loss of FGFs that usually occurs due to their adherence to the surface of storage containers was effectively prevented by inclusion of 0.1% CHAPS; 3) the recovery of FGFs after storage or dialysis was significantly enhanced by inclusion of 0.1% CHAPS; 4) CHAPS at lower concentrations than 0.1% does not interfere with amino acid analysis, except that Thr may be misled only when the ratio of protein/CHAPS is low; 5) amino acid sequence analysis was hardly disturbed by CHAPS up to 0.5%. These results indicate that CHAPS is useful as a stabilizing agent for various kinds of polypeptides capable of showing biological activity at a low concentration. Editor's statement Polypeptide growth factors and hormones, especially heparin-binding growth factors (FGF), are notoriously troublesome to handle in purified form at extremely low concentrations. This report demonstrates an interesting solution to this problem.     

A quantitative and selective chromatography method for determining coverages of multiple proteins on surfaces

Competitive protein adsorption plays a key role in the surface hemocompatibility of biological implants. We describe a quantitative chromatography method to measure the coverage of multiple proteins physisorbed to surfaces. In this method adsorbed proteins are displaced by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and then analyzed by high performance liquid chromatography to separate and quantify the individual proteins, in this case bovine serum albumin (BSA) and bovine fibrinogen (Fg). CHAPS displaced over 95% of the adsorbed proteins and was easily removed from solution by dialysis. This method was tested by measuring the coverage of BSA, 66 kDa, and Fg, 340 kDa, simultaneously adsorbed from solutions with concentration of 20 microg/ml, on bare and dextranized silicon. Relative to silicon, the dextranized surfaces were found to strongly inhibit protein adsorption, decreasing BSA and Fg coverages by 76 and 60%, respectively.     

CHAPS Solubilization and Functional Reconstitution of beta-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue

A method for the solubilization and reconstitution of red beet (1,3)-β-d-glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg²⁺ prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.     

Isotope-edited multidimensional NMR of calcineurin B in the presence of the non-deuterated detergent CHAPS

Summary At the concentration needed for NMR, the calcium-saturated form of calcineurin B dissolved in water shows resonance line widths that indicate aggregation of this protein. Although the line width or aggregation state can be influenced to some degree by temperature, pH, and salt concentrations, in the absence of detergent no conditions could be found where the protein behaved as a monomeric unit. In the presence of a 10- to 20-fold molar excess of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), resonance line widths were considerably narrower and were compatible with a protein of 25 kDa. The presence of the NMR signals of the non-deuterated CHAPS does not interfere with modern isotope-directed NMR studies as the signals from protons not attached to ¹⁵N or ¹³C are removed by isotope filtering and purge pulses.     

Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.     

The use of a zwitterionic detergent in two-dimensional gel electrophoresis of trout liver microsomes

CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-Propane sulfonate, a zwitterionic detergent, has been shown to exhibit superior membrane protein solubilizing characteristics as compared to nonionic detergents. Replacement of NP-40 with CHAPS in isoelectric focusing of rainbow trout liver microsomes has increased resolution markedly. The two-dimensional electrophoretic system described will allow effective resolution of up to 300 micrograms of crude microsomal protein. CHAPS exhibits no effect on the stability or type of pH gradient when compared to NP-40 during isoelectric focusing in the presence of urea.     

Assay of protein in the presence of high concentrations of sulfhydryl compounds

A simple procedure is described for removal of Triton X-100 from protein samples by adsorption of the detergent on a commercial copolymer in bead form. The concentration of detergent can be lowered to approximately 0.01% with no loss or dilution of protein.     

Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium

In order to maximize the myocardial proteome observed by two-dimensional gel electrophoresis (2-DE), the effect of (1) either an ionic or different zwitterionic detergents during tissue homogenization and (2) altering the "standard" detergent for isoelectric focusing (3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS)) to either the zwitterionic detergent amidosulfobetaine-14 (ASB-14) or N-decyl-N-N'-dimethyl-3-ammonio-1-propane sulfonate (SB3-10) was investigated. Sodium dodecyl sulfate was shown to be a superior detergent for extraction of proteins during homogenization of cardiac tissue compared to the detergents ASB-14, SB3-10 or CHAPS. Additionally, both ASB-14 and SB3-10 exhibited better extraction than CHAPS for distinct regions of two-dimensional gels. In most cases, the best combination of homogenization and focusing conditions did not involve the use of the same detergent. Specifically, it was found that the ability to mix homogenization and focusing conditions can allow one to obtain an optimum balance between the resolution and number of protein spots obtained in 2-DE analysis of cardiac tissue. An excellent initial combination of buffers to utilize for the general examination of cardiac proteins was determined to be initial homogenization in a buffer containing ASB-14 followed by focusing in a buffer containing CHAPS.     

Solubilization of UDPglucose-ceramide glucosyltransferase from the Golgi apparatus

The choice of a suitable detergent for solubilization of UDPglucose-ceramide glucosyltransferase from Golgi membranes has been investigated. Among the various classes of detergent, CHAPS, a zwitterionic detergent, was used as it produced a substantial activation of the enzyme activity. 30% of the enzyme activity and 50% of proteins were solubilized in the first attempts. Further experiments were conducted with addition of a second detergent, Zwittergent 3-14 which increased enzyme recovery to 45%. Lastly, reducing the concentrations of buffer and divalent cations Mn2+, Mg2+ and introducing glycerol (20%, v/v) allowed 80% of proteins to be solubilized together with 68% of the ceramide glucosyltransferase activity.     

Solubilization of Active Brain α1-Adrenoceptors by a Zwitterionic Detergent

Solubilization of rat brain alpha 1-adrenoceptors was performed by treatment with 6 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio] - 1 - propanesulfonate). The alpha 1-adrenoceptor antagonist [3H]prazosin was shown to bind reversibly and specifically to the soluble extract obtained after centrifugation at 150,000 X g for 1 h. Separation of the soluble [3H]prazosin-bound complexes was performed by the polyethylene glycol precipitation technique followed by filtration. A Scatchard plot of the concentration-dependent binding curve showed only one class of binding sites, with a high affinity for [3H]prazosin. Affinity of the solubilized receptors for the ligand increased as the CHAPS concentration in the assay medium decreased; the number of binding sites remained unchanged (approximately equal to 70 fmol/mg protein). This corresponds to a 30% recovery of original membrane sites. The solubilized receptors presented the same characteristics of specificity and stereospecificity as membrane alpha 1-adrenoceptors. Moreover, 150 mM NaCl was found to modulate the affinity of epinephrine for the [3H]prazosin-bound soluble complex, as previously described for membrane preparations. Thus, CHAPS appears to be a suitable detergent for solubilizing rat brain alpha 1-adrenoceptors and preserving their functional activities.     

Molecularly imprinted poly β-cyclodextrin polymer: Application in protein refolding

Regarding our previous report on refolding of alkaline phosphatase [Yazdanparast and Khodagholi, 2005 Arch. Biochem. Biophys] it was found that in spite of the anti-aggregatory effect of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitteronic detergent, the recovered activity was almost the same as the recovered activity obtained through the unassisted approach. The low recovery yield is probably due to the bulky groups of the detergent that interfere with its entrance into the small cavity of the stripping agent, cyclodextrin, implying that the stripping of detergent molecules from the detergent–protein complexes plays a major role in successful refolding processes. To improve the efficiency of CHAPS stripping, we evaluated, for the first time, the stripping potential of a molecular imprinting polymer designed to replace β-CD. In this approach, CHAPS was used as the template and the refolding of GuHCl denatured alkaline phosphatase was studied. Our results indicated that under the optimally developed refolding environment and similar to stripping by soluble β-CD, a refolding yield of 79% was obtained for denatured alkaline phosphatase using 20 mg/ml of the molecularly imprinted poly (β-CD) polymer. The major advantage of the new stripping agent, besides of its recycling option and ease of separation from the finished product, is its high potential of preventing aggregate formation. Based on these results, it seems that the new stripping strategy can constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble β-cyclodextrin.     

Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

A process to prepare a synthetic filter material containing nutrients for biofiltration

In this study, an optimal process to prepare a synthetic filter material (poly(vinyl alcohol) (PVA)/peat/KNO(3) composite bead) containing nutrients was developed for biofiltration. The optimal preparing condition was that each of the peat and PVA aqueous solutions contains 6.4 g KNO(3) and the nitrogen content in the boric and phosphate aqueous solutions must retain higher than 3.94 and 1.52 g N/l, respectively. The equilibrium amount of water-soluble nitrogen dissolved out of the prepared composite bead was between 7.95 and 8.21mg N/g dry solid. The path of water-soluble nitrogen dissolving out of the A-type bead was the water-soluble nitrogen dispersed in the peat phase initially diffused into the outer PVA phase and then it diffused out of the bead surface. And the path of water-soluble nitrogen dissolving out of the H-type bead was the water-soluble nitrogen dispersed in both the peat and PVA phases simultaneously diffused into the outer PVA phase and out of the bead surface, respectively. The microbial growth rate k(g) of the H-type composite bead was higher than that of the A-type composite bead approximately 1.09-1.58 times, and its value was between 0.100 and 0.417 day(-1) as the composite bead was immersed in 0-0.896 M KNO(3) solution. The maximum value of k(g) appeared at the composite bead immersed in 0.384 M KNO(3) solution and was higher than that of the compost by a factor approximately 1.49. The percentage of removed volatile organic compounds (VOCs) remained at more than 98% during the biofilter operating 230 days as the composite bead was immersed in KNO(3) aqueous solution before packing. This composite bed was without the further addition of nutrients during this operating period. It was proved that this composite bead was superior to the compost as a filter material.     

Developmentally regulated compartmentalization of adenylate cyclase in Dictyostelium discoideum

Adenylate cyclase of aggregation phase Dictyostelium discoideum is activated by extracellular adenosine 3', 5'-cyclic monophosphate (cAMP), and the cAMP synthesized is secreted. The distribution of the enzyme was determined in sucrose gradients loaded with whole cell lysates. Cell lysates prepared after 4.5 hr of starvation revealed membranes containing adenylate cyclase at 44% and 33% sucrose. The activity of the latter peak was detected in the presence of the detergent (CHAPS), 3-(3-cholamidopropyl) dimethylammonio-3-propanesulfonate, which inhibited the activity of the former to some extent. Adenylate cyclase activity of the 2 peaks differed with respect to solubility in CHAPS and their kinetics. The 44% sucrose region of the gradient contained the bulk of the plasma membranes, as judged by a cell surface glycoprotein marker (contact site A). The 33% peak is composed of small vesicular structures, as determined by electron microscopy. The distribution of adenylate cyclase activity detected in sucrose gradients shifted from the 33% to the 44% sucrose peak during development. In addition, the 44% peak became increasingly resistant to the inhibitory effect of CHAPS. Both changes were accelerated by extracellular cAMP, but only the latter was abolished when the production of endogenous cAMP was inhibited by caffeine. Pulsing cells with cAMP overcame the inhibitory effect of caffeine.     

High sensitivity identification of membrane proteins by MALDI TOF-MASS spectrometry using polystyrene beads

Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents.     

Fabrication and characterisation of a novel pellicular adsorbent customised for the effective fluidised bed adsorption of protein products

A dense pellicular solid matrix has been fabricated by coating 4% agarose gel on to dense zirconia-silica (ZS) spheres by water-in-oil emulsification. The agarose evenly laminated the ZS bead to a depth of 30 μm, and the resulting pellicular assembly was characterised by densities up to 2.39 g/mL and a mean particle diameter of 136 μm. In comparative fluidisation tests, the pellicular solid phase exhibited a two-fold greater flow velocity than commercial benchmark adsorbents necessary to achieve common values of bed expansion. Furthermore, the pellicular particles were characterised by improved qualities of chromatographic behaviour, particularly with respect to a three-fold increase in the apparent effective diffusivity of lysozyme within a pellicular assembly modified with Cibacron Blue 3GA. The properties of rapid protein adsorption/desorption were attributed to the physical design and pellicular deployment of the reactive surfaces in the solid phase. When combined with enhanced feedstock throughput, such practical advantages recommend the pellicular assembly as a base matrix for the selective recovery of protein products from complex, particulate feedstocks (whole fermentation broths, cell disruptates and biological extracts).     

Simultaneous reduction and alkylation of protein disulfides in a centrifugal ultrafiltration device prior to two-dimensional gel electrophoresis

Reduction and alkylation of protein disulfides prior to IEF, when performed directly in a centrifugal ultrafiltration device, provides an effective means of terminating the alkylation reaction, concentrating the proteins for analysis, and removing ionic impurities that interfere with IEF. When cells were lysed in "buffers" that support the activity of enzymes such as lysozyme and benzonase, the conductivity of the resulting lysate was an order of magnitude higher than when lysis was induced by chaotropic urea detergent solutions. Following reduction and alkylation, the conductivity of both lysates was lowered by ultrafiltration to the 0.1-0.2 mS/cm range in preparation for IEF. The detergent 3-(4-heptyl)phenyl 3-hydroxypropyl dimethylammonio propanesulfonate (C7BzO), which favors the solubilization of proteins, but which interferes with SDS equilibration and second dimension PAGE, was effectively removed by ultrafiltration and exchanged with CHAPS without measurable loss of protein. Disparate protein patterns of Rhodopseudomonas palustris lysates were revealed by two-dimensional gel electrophoresis depending on which reagent was used to induce cell lysis.     

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.     

Soluble and enzymatically stable (Na+ + K+)-ATPase from mammalian kidney consisting predominantly of protomer alpha beta-units. Preparation, assay and reconstitution of active Na+, K+ transport

Soluble (Na+ + K+)-ATPase consisting predominantly of alpha beta-units with Mr below 170 000 was prepared by incubating pure membrane-bound (Na+ + K+)-ATPase (35-48 mumol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). (Na+ + K+)-ATPase and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5-3, at which 50-70% of the membrane protein was solubilized. The soluble protomeric (Na+ + K+)-ATPase was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5-6, at the expense of partial inactivation, but (Na+ + K+)-ATPase and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, S20,w was 7.4 S for the fully active (Na+ + K+)-ATPase, 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000-170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active (alpha beta)2-dimers or (alpha beta)3-trimers with S20,w = 10-12 S and apparent molecular masses in the range 273 000-386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles.