Protein nanopores with covalently attached molecular adapters

Molecular adapters are crucial for the stochastic sensing of organic analytes with alpha-hemolysin (alphaHL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the alphaHL pore. In the present work, we have realized the controlled covalent attachment of a beta-cyclodextrin (betaCD) adapter in the two possible molecular orientations inside alphaHL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the betaCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alphaHL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.     

Copper and iron interactions with angiotensin-converting enzyme inhibitors. A study by fast-atom bombardment tandem mass spectrometry

Fast atom bombardment, combined with high-energy collision-induced tandem mass spectrometry, has been used to investigate gas-phase metal-ion interactions with captopril, enalaprilat and lisinopril, all angiotensin-converting enzyme inhibitors.Suggestions for the location of metal-binding sites are presented. For captopril, metal binding occurs most likely at both the sulphur and the nitrogen atom. For enalaprilat and lisinopril, binding preferably occurs at the amine nitrogen. Copyright 1999 John Wiley & Sons, Ltd.     

Synthetic porphyrins at interfaces; photosensitization and related reactions of atropisomers of o-substituted tetraphenylporphine derivatives in films, reversed micelles and membranes

Abstract— Previous studies from our laboratories and elsewhere have shown that amides between fatty acids and the synthetic α, α, α, α -tetra (o-aminophenyl)-porphyrins exhibit good surfactant properties which facilitate their incorporation into structured assemblies characterized by hydrophobic-hydrophilic compartmentalization. This paper will focus on a number of aspects of our studies of these porphyrins at different interfaces. The α, α, α, α (4,0) isomers are readily incorporated into Langmuir-Blodgett films as either free base or metal complexes. Studies of assemblies containing free base and palladium (II) complexes have been carried out in which the porphyrin is irradiated in the presence of oxygen and nonexcited but oxidizable substrates. Much of the reactivity observed can be attributed to ¹O₂* generation. These studies reveal the migration range and reactivity of activated oxygen in a structure related to biomem-branes. Several of these synthetic porphyrins have also been examined in cell suspensions and in synthetic reversed micelle solutions. Studies in the former have shown that the porphyrins can mediate the photoinactivation of several enzymes located inside and within the mitochondrial membrane in tumor cells extracted from rats. They are found to compare favorably to hematoporphyrin derivative in effectiveness. Studies of the same porphyrins in their reactivity towards copper ion incorporation in anionic reversed micelles indicate striking rate differences which can be interpreted in part to structural variations between the porphyrins as well as to their orientation at the hydrophobic-hydrophilic interface.     

Formation and decay of doubly charged ions in n-alkanes from methane to triacontane

Coincidence techniques were used to study dissociative double ionization of selected n-alkanes from methane to triacontane (C₃₀H₆₂) and of the hexane isomers. Following photoionization at 40.8 eV, both covalent and coulombic dissociations of the molecular dications take place. The main decay route of doubly charged alkanes larger than butane is fast charge separation followed by secondary dissociation of energetic singly charged primary ions. A simulation based on quasi-equilibrium theory and the spectra of the isomers confirm this breakdown mechanism for hexane.