Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.     

Theoretical study of flow,pressure and solute concentration in double membrane systems

A model system consisting of two rigidly held membranes in series was investigated through the application of the Kedem and Katchalsky thermodynamic single membrane flow equations. This analysis results in predictions of the steady state flow properties as well as values for the solute concentration and pressure of the internal compartment when the system is under the influence of a constant solute concentration or hydrostatic pressure gradient. It is demonstrated that although the flow properties and internal compartment pressure are complicated functions of the membrane permeability coefficients and driving gradient across the system, the relationships are greatly simplified by the explicit appearance of the internal compartment steady state solute concentration in the equations. It is shown that the steady state volume flow rate depends on the absolute value of the solute concentration in the external compartments, as well as the solute concentration gradient across the system. The properties of non-linear dependence of volume flow on concentration gradient, and rectification of volume flow are discussed and shown to be independent properties of the system. For the system under the influence of a solute concentration gradient, the internal compartment pressure can be greater or less than the ambient pressure, and depends mainly on the order in which the membranes are encountered by the volume flow. These properties are qualitatively correlated with certain available experimental observations in biological systems.