We present in this paper that porous silicon can be used as a large surface area matrix as well as the transducer of biomolecular interactions. We report the fabrication of heavily doped p-type porous silicon with pore diameters that can be tuned, depending on the etching condition, from approximately 5 to 1200 nm. The structure and porosity of the matrixes were characterized by scanning force microscopy (SFM) and scanning electron microscopy (SEM), Brunnauer-Emmett-Teller nitrogen adsorption isotherms, and reflectance interference spectroscopy. The thin porous silicon layers are transparent to the visible region of the reflectance spectra due to their high porosity (80-90%) and are smooth enough to produce Fabry-Perot fringe patterns upon white light illumination. Porous silicon matrixes were modified by ozone oxidation, functionalized in the presence of (2-pyridyldithiopropionamidobutyl)dimethylmethoxysilane, reduced to unmask the sulfhydryl functionalities, and coupled to biotin through a disulfide-bond-forming reaction. Such functionalized matrixes display considerable stability against oxidation and corrosion in aqueous media and were used to evaluate the utility of porous silicon in biosensing. The streptavidin-biotin interactions on the surface of porous silicon could be monitored by the changes in the effective optical thickness calculated from the observed shifts in the Fabry-Perot fringe pattern caused by the change in the refractive index of the medium upon protein-ligand binding. Porous silicon thus combines the properties of a mechanically and chemically stable high surface area matrix with the function of an optical transducer and as such may find utility in the fabrication of biosensor devices.

Convenient methodology for preparation and conjugation of the protein-cutting iron chelate iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) is given. This formulation of the reagent can be handled in a manner analogous to many other protein-labeling reagents, such as fluorescent probes or cross-linkers. By taking advantage of the recently discovered peptide hydrolysis reaction, the chelate may be tethered to a single site (e.g., a cysteine side chain) and used to map its proximity to individual peptide bonds by automated Edman sequencing of the protein fragments produced. The method is illustrated by conjugation of Fe-BABE to the carboxy terminal domain (amino acid residues 234-329) of the Escherichia coli RNA polymerase alpha subunit. The molecular mass of the protein conjugate was confirmed by electrospray ionization mass spectrometry.

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (a!2313') and the holoenzyme (core + .70) shows that absence of the Or70 subunit is associated with the appearance of several cleavage sites on the subunits fJ (within 10 residues of sequence positions 745, 764, 795, and 812) and 3' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near 13 residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when cr70 binds. No differences are observed for the a subunit.Gene transcription in living cells is a complex process, with large numbers of protein factors involved in selecting the correct initiation site on DNA and initiating and carrying out RNA synthesis to the appropriate end. The proteins responsible for transcription in both prokaryotic and eukaryotic cells are actively being identified and their functions explored (1-8).Bacterial RNA polymerase is a well-studied example. It is composed of five protein subunits (Ct2f1'o), of which one (o-) is a member of a group of related proteins that convey different DNA-binding specificities. The most common a subunit in Escherichia coli has a mass of 70 kDa and is designated a70.It has long been known that oa70 binds reversibly to the at23' core enzyme (9-11), but the actual binding site has been difficult to identify. This problem has been addressed by chemical cross-linking (12-14) and by molecular genetics (15), but identification of the particular amino acid residues on the core subunits involved in oJ7() binding remains a challenge.

A novel experiment has been used to show proximity relationships between sites on the surface of the cytochrome bd quinol oxidase of Escherichia coli. The artificial protease iron (S)-1-[p-(bromoacetamido)benzyl]-EDTA (Fe--BABE) was conjugated to selected reactive cysteines placed in subunit I or subunit II, with the aim of identifying amino acid residues within approximately 12 A of each site of attachment. The protease was activated with H2O2 and ascorbate for a few seconds, and hydrolysis products were isolated and analyzed by N-terminal sequencing. Among other results, we found that residue 39 of subunit II is near residue 255 of subunit I in the putative quinone-binding domain (Q loop) of the oxidase. Since this technique is insensitive to the nature of the amino acid side chains, it should prove generally valuable in revealing spatial relationships both within and between subunits in complex proteins where high-resolution structural information is not available.