Abstract
In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB14, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7-11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0-4°C, at 200 V in 6-cm gels of 5% T 15% CBis with 0.1 n H2SO4 and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 201-208 |
| Number of pages | 8 |
| Journal | Analytical Biochemistry |
| Volume | 95 |
| Issue number | 1 |
| DOIs | |
| State | Published - 1979 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents. / Hjelmeland, Leonard M; Nebert, Daniel W.; Chrambach, Andreas.
In: Analytical Biochemistry, Vol. 95, No. 1, 1979, p. 201-208.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents
AU - Hjelmeland, Leonard M
AU - Nebert, Daniel W.
AU - Chrambach, Andreas
PY - 1979
Y1 - 1979
N2 - In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB14, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7-11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0-4°C, at 200 V in 6-cm gels of 5% T 15% CBis with 0.1 n H2SO4 and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.
AB - In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB14, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7-11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0-4°C, at 200 V in 6-cm gels of 5% T 15% CBis with 0.1 n H2SO4 and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.
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UR - http://www.scopus.com/inward/citedby.url?scp=0018398661&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(79)90206-9
DO - 10.1016/0003-2697(79)90206-9
M3 - Article
C2 - 495956
AN - SCOPUS:0018398661
VL - 95
SP - 201
EP - 208
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -