Thymosin p4 (Tp4), a peptide of 43 amino acids, binds to actin monomers and inhibits filament formation. In preparations of Tp4 from bovine lung tissue, the peptide is accompanied by a derivative in which the methionine residue in position 6 is replaced by its sulfoxide. Tp4 sulfoxide inhibits actin polymerization to an extent approximately 20-times less than Tp4. While an equimolar amount of Tp4 prevented actin polymerization almost completely, polymerization with the corresponding amount of the sulfoxide proceeded in a manner similar to that of pure actin, except for a slight retardation. We showed that the decrease in the inhibitory activity is reflected by a 20-times lower affinity to actin. Interestingly, under non-polymerizing conditions, the affinity of Tp4 sulfoxide for actin is as high as that of Tp4 (approximately 1 pM). In accordance with this, no differences were found between Tp4 and the sulfoxide in cross-linking experiments with the monomer, where both forms of the peptide yielded similar amounts of a 47-kDa band representing conjugates of actin and P-thymosin, as proved by Western-blotting analysis. Likewise, both, Tp4 and the sulfoxide retarded the exchange of G-actin-bound nucleotide to similar extents. Although the sulfoxide is presumably a product of autoxidation, it is attractive to speculate that oxidation of the methionine residue in Tp4 may represent a regulatory switch for starting filament formation in non-muscle cells.

A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immuno-histochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri- and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.