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Neo carzinostatin chromophore purification of the major active form and characterization of its spectral and biological properties

Neo carzinostatin chromophore purification of the major active form and characterization of its spectral and biological properties

Biochemistry 20(19): 5602-5608

The nonprotein chromophore of neocarzinostatin (NCS-Chrom), which possesses the full biological activity [antitumor] of native NCS, can be separated by high-performance liquid chromatography into 2 components, A and B, with 90 and 10%, respectively, of the total UV-absorption and in vitro DNA scission activity. Each component possesses in vitro DNA scission activity equal to NCS, as does a 3rd component, C, which is present only when NCS is extracted with nonacidic methanol and appears to be derived from A. NCS-Chrom A possesses in vivo activity equal to NCS; however, B and C have about 5 and 80%, respectively, of the activity of NCS for inhibition of HeLa [human cervical carcinoma] cell growth. NCS-Chrom A possesses characteristic absorption, fluorescence, circular dichroism (CD), and magnetic circular dichroism (MCD) spectra. The spectral properties of B and C are similar to those of A. These spectral properties cannot be attributed solely to the presence of the known naphthoate component of NCS but are due to the presence of an additional conjugated component (as yet unidentified) in the chromophore. NCS-Chrom D, an inactive minor component of NCS-Chrom preparations, can be generated by hydrolysis of each of the 3 active components at pH 8. It possesses a very different CD spectrum and, in contrast to NCS-Chrom, possesses no MCD, indicating a major change in or loss of the naphthoic acid residue. NCS-Chrom D is the 490-nm fluorescent product generated when NCS-Chrom spontaneously looses biological activity in aqueous pH 8 buffers. It is the source of the 490-nm fluorescence in clinical NCS. The optical activity of the biologically active NCS-Chrom accounts for the Cotton effects previously observed for native NCS. Changes in both the absorption and circular dichroism spectra of NCS-Chrom A (and B and C) occur on binding to apo-NCS, generating spectra identical with those of native NCS and providing stoichiometry of the association process. Complex formation between NCS-Chrom and DNA is also detected by CD, with the maximal effect being observed at low molar ratios of NCS-Chrom to DNA. Hypochromicity between 290 and 330 nm and a bathochromic shift centered near 365 nm occur upon binding of NCS-Chrom to apo-NCS and to DNA, raising the possibility that similar modifications in the electronic structure of the NCS-Chrom result from both interactions.

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Accession: 005962561

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PMID: 6457635

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