Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

Abstract

A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.

Fig. 1.

Rates of thermal inactivation and aggregation of BfAChE. Samples of BfAChE (15 μM) in buffer 1 were inactivated at 52°C. Aliquots were withdrawn at appropriate times for assay of enzymic activity or for assessment of monomeric content by PAGE under nondenaturing conditions. (A) Filled circles: residual activity; open triangles: percentage of monomer estimated by scanning of the acrylamide gel. (B) Samples were added to ice-cold sample buffer, frozen, and thawed immediately prior to loading of the acrylamide gel. PAGE under nondenaturing conditions was performed on 5% acrylamide gels as described under Materials and Methods. Each lane was loaded with 10 μg of protein. Staining was with Coomassie blue.

Fig. 2.

Analytical sucrose gradient centrifugation of native and heat-denatured BfAChE. Sucrose gradient centrifugation was performed on 5–20% linear sucrose gradients in buffer 1, essentially as described previously (24). Open squares: native BfAChE; filled circles: BfAChE denatured by heating for 90 sec at 53°C. Native enzyme was assayed by its enzymic activity, and heat-denatured enzyme using intrinsic fluorescence. Catalase (11.3 S), and A₁₂ Electrophorus AChE (18 S), served as internal standards. Centrifugation was performed using an SW40Ti rotor in a Beckman L7–55 ultracentrifuge at 28,000 rpm for 15 h.

Fig. 3.

Crosslinking of native and heat-denatured BfAChE by hypericin as demonstrated by SDS-PAGE. Electrophoresis was performed as described previously, but under nonreducing conditions, using a 3.5–15% acrylamide gradient. Lane 1, native TcAChE dimer (ca. 130 kD). Lane 2, native BfAChE monomer. Lane 3, native BfAChE + hypericin, irradiation for 10 min. Lanes 4–6, heat-denatured BfAChE + hypericin, irradiation for 10 min; heat denaturation was performed for 2, 5, and 10 min, respectively, at 50°C, in buffer 1. Lane M, high molecular weight markers.

Fig. 4.

Concentration-dependence of the rate of disappearance of monomeric species of BfAChE. The rate of disappearance of monomer, at 53°C, was monitored by PAGE under nondenaturing conditions (see legend to Fig. 1 ▶), and the data were plotted as ln [monomer] versus time. Open circles: 2.5 μM; filled circles: 5 μM; open triangles: 10 μM.

Fig. 5.

Kinetics of thermal inactivation of BfAChE inside reverse Brij 30 micelles and in aqueous solution in the presence of normal Brij 30 micelles. Inactivation was performed at 45°C. In both cases, the enzyme concentration was 10 μM. Open circles: BfAChE in reverse Brij 30 micelles, w₀ = 15; filled circles: BfAChE in solution in buffer 1 containing 0.1% Brij 30. Monoexponentional fits were obtained, with correlation coefficients of 0.99 and 0.97, respectively, for reverse and normal micelles.

Fig. 6.

Kinetics of thermal inactivation of BfAChE inside LUV. Samples of BfAChE entrapped in LUV were incubated at 51°C. Filled circles: protein/vesicle ratio, 1:1; open circles: protein/vesicle ratio, 10:1. Monoexponential fits were obtained with correlation coefficients of 0.99 and 1.00, respectively.

Fig. 7.

Normalized intrinsic fluorescence emission spectra of thermally inactivated BfAChE in reverse micelles and in aqueous solution. BfAChE (2 μM), either entrapped in reverse micelles, w₀ = 10 (A), or in aqueous solution (B), was inactivated at 51°C for 15 min. —, native BfAChE; –––, thermally inactivated BfAChE.

Fig. 8.

CD spectra of thermally inactivated BfAChE in reverse micelles and in aqueous solution. BfAChE (8 μM), either entrapped in reverse micelles, w₀ = 15 or in aqueous solution, was inactivated at 51°C for 15 min. 1. Native BfAChE; 2. Native BfAChE in reverse micelles; 3. Thermally inactivated BfAChE in aqueous solution; 4. Thermally inactivated BfAChE in reverse micelles.

Fig. 9.

Time course of refolding of BfAChE unfolded in 5 M Gdn HCl. Unfolding and refolding were as described under Materials and Methods. Refolding was performed in the presence (filled circles) or absence (open circles) of molecular chaperones.

Fig. 10.

Time course of reactivation of BfAChE after thermal inactivation. Samples of BfAChE in buffer 1, or entrapped inside reverse micelles, w₀ = 10, were inactivated at 48°C. When residual activity had reached ∼4.5%, samples were transferred to 4°C, and then slowly reheated to 34°C at a rate of ∼7.5°/h. Open circles: 0.6 μM BfAChE in aqueous buffer; filled circles: entrapped in reverse micelles.

Scheme 1.

Scheme 2.