A simple, stable, ultrasensitive dual-capillary dual-bicell (DCDB) microinterferometic backscattering detection (MIBD) system was developed. In DCDB MIBD, a He-Ne laser beam passes through a half-wave plate onto the cross section of two capillaries, one for reference and another for sensing analyte. The position of the backscattered fringe from each capillary, which are in proximity or essentially identical thermal environments, was detected with matched bicell photodetectors. The configuration was found to effectively compensate for thermal drift, which is normally the major source of noise in refractive index (RI) detection systems. It is shown that passive environmental compensation leads to greatly enhanced signal in nanoscale refractometry preformed by MIBD. An order of magnitude improvement in detection limits over single channel configurations is possible. Performance reaches the 10(-9) RIU level for like solvents in the presence in extremely large thermally induced RI gradients. At this level of detectability, DCDB MIBD could facilitate nanoliter-volume, femtomole-level universal detection in applications ranging from mu-HPLC and on-chip CE to scanning microcalorimetry.

Mutations in the ATP-binding cassette (ABC) transporters ABCG5 and ABCG8 lead to sitosterolemia, a disorder characterized by sterol accumulation and premature atherosclerosis. ABCG5 and ABCG8 are both half-size transporters that have been proposed to function as heterodimers in vivo. We have expressed the recombinant human ABCG5 and ABCG8 genes in the yeast Pichia pastoris and purified the proteins to near homogeneity. Purified ABCG5 and ABCG8 had very low ATPase activities (<5 nmol min(-)(1) mg(-)(1)), suggesting that expression of ABCG5 or ABCG8 alone yielded nonfunctional transporters. Coexpression of the two genes in P. pastoris greatly increased the yield of pure proteins, indicating that the two transporters stabilize each other during expression and purification. Copurified ABCG5/G8 displayed low but significant ATPase activity with a V(max) of approximately 15 nmol min(-)(1) mg(-)(1). The ATPase activity was not stimulated by sterols. The catalytic activity of copurified ABCG5/G8 was characterized in detail, demonstrating low affinity for MgATP, a preference for Mg as a metal cofactor and ATP as a hydrolyzed substrate, and a pH optimum near 8.0. AlFx and BeFx inhibited MgATP hydrolysis by specific trapping of nucleotides in the ABCG5/G8 proteins. Furthermore, ABCG5/G8 eluted as a dimer on gel filtration columns. The data suggest that the hetero-dimer is the catalytically active species, and likely the active species in vivo.