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Studies on the efficiency of several selected nonionic detergents in solubilizing acetylcholinesterase from bovine erythrocyte membrane

University of British Columbia

In the present report, several nonionic and zwitterionic detergents have been examined to determine their efficiency in solubilizing acetylcholinesterase (EC 3.1.1.7) (AChE) from bovine erythrocyte membrane. The objective of this study ultimately was to choose one or more detergents that would be suitable for purifying the enzyme from the erythrocyte membrane in a form representing the enzyme in its native environment. In the present study, AChE activity was retained after solubilization of bovine erythrocytes with octyl-β-D-glucoside (30-100 mg/ml). The most effective solubilization occurred at detergent concentrations more than ten times higher than the critical micelle concentration (cmc). Enzyme inactivation was apparent at a still higher concentration (250 mg/ml). The octylglucoside-solubilized enzyme lost all its activity when it was passed through a hydrophobic polystyrene resin, Bio Beads SM-2. Tweens were found to be generally not very effective in solubilizing bovine erythrocyte AChE, requiring concentrations some 17,000 to 77,000 times greater than their cmc for maximum effect. At a fixed concentration of 1000 mg/ml, Tween 20 solubilized more AChE than Tween 40, which in turn solubilized more AChE than Tween 60. Thus, as the saturated alkyl chain length of the acid portion of the molecule is increased, there is a decrease in AChE solubilizing capacity. Tween 80, whose hydrophobicity is less than that of Tween 60 due to the presence of the unsaturated double bond, has a solubilizing capacity similar to that of Tween 20. Almost all detergents that solubilize membranes efficiently have a HLB ranging from 12.5 to 14.5. In the present study, Tween 20, with the highest HLB of 16.7, was found to be the most efficient solubilizer, while Tween 60, with the lowest HLB of 14.9, was found to be the least efficient solubilizer. Tween 80, with an HLB of 15.0, appears anamolous. This may be because Tween 80 contains a more fluid unsaturated chain, in contrast to the other Tweens. Increase in micelle size occurs with increasing hydrocarbon chain length. The solubilizing ability of nonionic detergents generally decreases as the saturated alkyl chain length is increased above 16 carbon atoms. In the study on the Zwit-tergent series of detergents, as the cmc of the detergent increases from the least polar Zwittergent 316 to Zwittergent 310, a decrease in enzyme activity was detected in the supernatant. This behavior is in contrast to the Tween series. Thus, the longer chain length compound, Zwittergent 316, was the most efficient detergent in the series for the recovery of active enzyme activity in the supernatant. The amount of enzyme activity solubilized by Z 316 increased with increasing concentration up to a 300:1 ratio of detergent concentration to cmc. By contrast, the shorter chain Zwittergents were most effective at concentrations at or below their cmc, suggesting that solubilization occurred by a different mechanism to Z 316. The relationship between chain length and solubilization of AChE in an active form could also be examined in the Lubrol class of detergents. Lubrol PX has a shorter polyoxyethylene chain (9-10) than Lubrol WX, which averages 17 polyoxyethylene chains. While both detergents solubilize similar amounts of protein from bovine erythrocytes, both at 100 mg/ml and 500 mg/ml, Lubrol PX was more effective than Lubrol WX in maintaining the AChE active in the 100,000 x g-soluble form. Higher concentrations of both Lubrol WX and Lubrol PX (500 mg/ml) decreased the overall enzyme recovery. Triton X-100 was found to be one of the most efficient of the detergents examined. The high efficiency is consistent with its low cmc (0.16 mg/ml), its large micelle size and the favorable HLB. However, Triton X-100 was not one of the most effective detergents for the purification of the enzyme, as considerably higher specific activities, after affinity chromatography, were obtained with Lubrol PX and Tween 20, while Z 316 was the least effective of the compounds studied. Determination of the sedimentation coefficient of bovine erythrocyte AChE purified by affinity chromatography following solubilization by various detergents was used as an indication of the molecular structure of the enzyme solubilized by each detergent. Solubilization with Lubrol PX, Tween 20 and Triton X-100 yielded AChE corresponding to S values of 6.8 S to 7A S, corresponding to enzyme dimers. AChE solubilized and purified following Zwittergent 316 behaved differently to the enzyme solubilized in the other detergents. In addition to the dimeric 7.1 S form, a large amount of enzyme activity appeared in an aggregated form. Functional characteristics of the various detergent-purified AChE preparations were assessed by Arrhenius plots. A break in the Arrhenius plot at around 16°C was observed in AChE purified with Tween 20 and Lubrol PX. Furthermore, the break was abolished after high salt treatment (1.8 M NaCl). From previous work high salt treatment is known to cause functional but not physical dissociation of a tightly bound fraction of lipid, probably cardiolipin, from the enzyme, as the resulting enzyme behaves as a monomer instead of a dimer by irradiation inactivation or target size analysis. Thus, the non-linear Arrhenius plot appears to be independent of the detergent used for extracting the enzyme species, and therefore represents an intrinsic property of the enzyme itself.

Text

2010-07-08

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http://hdl.handle.net/2429/26206

Pharmaceutical Sciences

University of British Columbia

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