Evaluation of the efficacy of zwitterionic dodecyl carboxybetaine surfactants for the extraction and the separation of mycoplasma membrane protein antigens.
The ability to extract mycoplasma membrane protein antigens using the alkyl carboxybetaine surfactants (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC = 4.3 mM) and (N-dodecyl-N,N-dimethylammonio)undecanoate (DDMAU, CMC = 0.13 mM) was assessed by protein titration and SDS-PAGE analysis. The maximum yields of membrane protein solubilization ranged from 20 to 90%, depending upon both the mycoplasma membrane investigated and the surfactant used. In five of six cases, the extraction was optimal for surfactant concentrations of ca. 25 mM. DDMAB displayed a higher efficiency in membrane protein extraction. The order of efficiency for both surfactants was Spiroplasma melliferum > Acholaplasma laidlawii > Mycoplasma gallisepticum. In contrast, DDMAU proved much more selective. The order of selectivity was M. gallisepticum > S. melliferum > A. laidlawii. The highest selectivity was recorded for the major proteins p67 and spiralin of M. gallisepticum and S. melliferum, respectively. For p67, notably, DDMAU proved superior to 10 other surfactants. Dot immunobinding and crossed immunoelectrophoresis analyses showed that both dodecyl carboxybetaines were suitable as membrane protein-solubilizing agents in immunological techniques. Furthermore, these surfactants did not exhibit effects adverse to the activity of A. laidlawii membrane NADH oxidase. One promising application of DDMAU is the separation of membrane proteins by ion-exchange HPLC as illustrated by the good resolution of M. gallisepticum membrane proteins and purification of p67 to almost homogeneity. These data show that dodecyl carboxybetaine surfactants are useful for the extraction of mycoplasma membrane antigens under mild conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Brenner
C
C
Département Membranes et Osmorégulation, CNRS URA 256, Université de Rennes I, France.
Jan
G
G
Chevalier
Y
Y
Wróblewski
H
H
D000942:Antigens, Bacterial / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002087:Butyrates; D002852:Chromatography, Ion Exchange / Q000379:methods; D005069:Evaluation Studies as Topic; D005227:Fatty Acids; D015151:Immunoblotting; D007477:Ions; D009097:Multienzyme Complexes / Q000378:metabolism; D009174:Mycoplasma / Q000737:chemistry*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents
C093272:(N-dodecyl-N,N-dimethylammonio)butyric acid; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002087:Butyrates; D005227:Fatty Acids; D007477:Ions; D009097:Multienzyme Complexes; D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents; C093274:(N-dodecyl-N,N-dimethylammonio)undecanoic acid; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases