Capillary electrophoresis of closely related intrinsic thylakoid membrane proteins of the photosystem II light‐harvesting complex (LHC II)

Abstract

The electrophoretic migration behavior of three closely related hydrophobic intrinsic membrane proteins of the photosystem II light-harvesting complex (LHC II) was investigated in free solution capillary electrophoresis at pH 8.0-10 with running electrolyte solutions containing either anionic, zwitter-ionic or nonionic detergents. The complete and repeatable separation of these proteins was accomplished with a running electrolyte solution of 25 mM Tris/192 mM glycine, pH 8.8, containing either sodium dodecyl sulfate or n-octyl beta-D-glucopyranoside at concentration up to 5.0 and 7.0 mM, respectively. Migration times and resolution of the individual LHC II intrinsic membrane proteins were sensitive to the type of detergent. The effect of detergent concentration on the electrophoretic behavior of the LHC II proteins was also investigated. Electroelution of the LHC II components separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to isolate these intrinsic membrane proteins, which were then injected onto the capillary electrophoresis system for peak identification.

High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins

“…16 This is particularly true for thylakoid membranes, where the complete solubilization of intrinsic proteins normally requires strong detergent, which can interfere with biochemical assays. 17 A recent study reported the rapid resolution of the protein components of LHCII by capillary electrophoresis (CZE) 18 and reversed phase HPLC. 19,20 The present study was undertaken to obtain the resolution of all the protein components of PSI from spinach and their identification using a reversed phase HPLC column coupling the HPLC with a triple quadrupole mass spectrometer equipped with an electrospray ion source.…”

Microseparation of membrane proteins

“…Previously, Zolla et al [61] reported complete separation of the three intrinsic membrane proteins of the photosystem II light-harvesting chlorophyll -protein complex of higher plant thylakoid membranes (photosynthetic membranes) by CZE using uncoated fused-silica capillaries and running electrolyte solutions containing either an anionic SDS or nonionic octyl-b-D-glucopyranoside detergent.…”

Characterization of the transmembrane serine receptor by capillary zone electrophoresis

“…However, the routine use of urea is not appropriate for CZE protein separations since extra peaks and/or multiple peaks can appear due to the denaturing action of urea. Less harshly denaturing surfactant compounds, like SDS, have been used with some success to solubilize protein samples [12,21,24,27,28]. As Tsr had originally been solubilized in 0.5 % SDS (w/v) following purification, a matching level of SDS was included in the tetraborate run buffer at pH 9 and the sample was re-analyzed under the previous electrophoretic conditions.…”

“…Several approaches have been developed to reduce protein adsorption in CZE including the use of run buffers at pH extremes [4,6,8] or at high ionic strengths [13][14][15] or hydrophilic or hydrophobic run buffers serving to modify dynamically the capillary surface [4,10,13,[15][16][17][18][19][20]. Low concentrations of cationic [21][22][23][24] or non-ionic [12,21,25] or zwitterionic [22,26] surfactants act by binding or masking the silanol groups on the capillary wall. Other reports have suggested that anionic surfactants, like sodium dodecyl sulfate (SDS) [21,24,27,28] can lead to improved peak shapes and reduced wall adsorption for proteins by binding to their positive sites, thereby reducing interaction with the capillary wall.…”

“…CZE in uncoated capillaries has been applied extensively to soluble or partially soluble proteins [1-11], but very infrequently for intrinsic membrane proteins which are insoluble in the common aqueous CZE run buffers [1,12] based upon phosphate, borate, acetate, citrate or biological buffers such as CAPS and MES. The most important problem is associated with the adsorption of the proteins onto the fused-silica capillary wall which leads to poor peak shapes, low resolution, incomplete recoveries and irreproducible separations.…”