Crosslinking transcription factors to their recognition sequences with PtII complexes.

We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.     

A diagonal electrophoretic method for selective purification of methionine peptides

1. A method is described that selectively purifies methionine peptides from enzymic digests of a protein. The peptides, after paper electrophoresis, are treated on paper with iodoacetamide at acid pH. This specifically converts methionine residues into their sulphonium salts. When the paper is submitted to electrophoresis at right angles to the original direction, the carbamoylmethylmethionine peptides emerge from an undifferentiated diagonal. 2. Heating at neutral pH converts carbamoylmethylmethionine into homoserine and thereby specifically cleaves the peptides. 3. The effect of the modifications on amino acid composition and sequence analyses of the peptides was studied. 4. When the method was applied to a tryptic digest of S-aminoethyl-chymotrypsinogen A, two peptides were selectively purified that had the expected amino acid sequence.     

Kallikrein and kallikrein-like proteinases: purification and determination by chromatographic and electrophoretic methods

Kallikreins and kallikrein-like enzymes make up a family of serine proteinases present in tissues and body fluids of mammals and in some snake venoms. This review deals with the procedures of purification, detection and determination of these enzymes by chromatographic and electrophoretic methods. The procedures are reported in tables, described and discussed with the aim of illustrating the state-of-the-art of research in the field.     

Application of electrophoretic methods for detection of protein-porphyrin complexes

Simple methods for detection and isolation of protein-porphyrin complexes were elaborated in our laboratory. They are based on the separation of protein-porphyrin complexes in native polyacrylamide gel and measurement of their fluorescence, with the use of two detection systems: the commercially available Gel Doc(TM) 2000 system, and a system specially designed for the purpose of these investigations, concerning protein-porphyrin interactions. The fluorescent complexes can be electro-transferred from the gel onto PVDF membrane, eluted and analyzed in order to identify the protein interacting with porphyrins.     

Histone electrophoretic pattern in the characterization of synaptonemal complexes

The presence of a stoechiometric electrophoresis pattern of histones in carefully isolated synaptonemal complexes is reported. The use of this pattern is suggested as an internal standard of synaptonemal complex purification, in addition to the more generally used electron micrographs. This is especially useful in experiments leading to the characterization of the protein components of SCs. The use of mice of an age at which pachytenes predominate (90%) in the prophase-cell population is also advantageous to improve the final yield in synaptonemal complexes.