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PubMed1986

Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme.

Human airway lysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-1 at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteristic of the peptide backbone (e.g., beta-sheet; C alpha-C, C alpha-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% alpha-helix, 25% and 20% beta-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.

Amino Acids↗

PubMed1982

Interaction of adriamycin with DNA as studied by resonance Raman spectroscopy.

Raman and resonance Raman spectra of the complex DNA-adriamycin in aqueous solution have been recorded and analysed. Calf thymus DNA was used and it is found that in the complex DNA-adriamycin the chromophore of adriamycin is intercalated in the GC sequences. The substituents on the rings give hydrogen bonding interactions with the base pairs above and below the intercalation site. It is suggested from the Raman and resonance Raman spectral modifications that the phenolic groups of the chromophore are involved in the drug-DNA intercalation, in addition to pi-pi, hydroxyl and amino group interactions.

Animals↗

PubMedDate not supplied

Expression, purification and DNA-cleavage activity of recombinant 68-kDa human topoisomerase I-target for antitumor drugs.

The gene encoding human DNA topoisomerase (topo) I, the target of numerous anticancer drugs, has been subcloned into bacterial, yeast and baculovirus-based expression systems in attempts to overexpress the enzyme for extensive structural and functional characterisation. Expression in E.coli produced a protein which was not suitable for structural studies. Expression in the yeast system was more successful enabling the enzyme to be purified and characterised. However, the resulting yield was modest for our requirements and the full-length protein was found to be susceptible to proteolysis when expressed in this system. As it is known that topo I from human placental tissue contains significant quantities of a 68kDa proteolytic fragment which retains both DNA relaxation and cleavage activity, we have isolated this fragment and shown by N-terminal sequence analysis that it starts at Lysine-191. This information was used to construct vectors which direct the overexpression of this fragment in baculovirus infected insect cells. The recombinant protein has been purified to homogeneity in a yield of 5-10mg/l of cell culture. The fragment is stable and retains all of the DNA driving activities of the intact enzyme. We have characterised the interactions of the topo I fragment with synthetic DNA substrates and identified oligonucleotides and conditions that allow covalent complexes between 68kDa topo I and DNA to be formed with high efficiency and in large quantity. A flow linear dichroism technique has been further developed and applied for real-time monitoring of supercoiled (sc) DNA relaxation by the enzyme and for comparative analysis of inhibition of 68kDa topo I by camptothecin (CPT).

Animals↗