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A highly effective method consisting of two affinity chromatography steps and ion-exchange and gelfiltration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg2+-dependent single-strand multiple nicking of the sub-strate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (KmS = 43 nM) and considerable catalytic efficiency (k8caPp/KbS = 0.32 min-l'nM'1) of the reaction. Antibodies able to catalyze a variety of chemical transformations were developed in the last decade, using the strategy of raising antibodies to haptens that resemble the transition state of reactions, as was suggested by Jencks (1) (for review see refs. 2 and 3). An increasing number of autoantibodies with catalytic activity toward natural substrates (4-6), as well as antiidiotypic antibodies exhibiting a catalytic function (7), have also been described recently. To date, however, the antibodymediated catalysis is usually characterized by relatively low rate enhancements, indicating the existence of at least two very important problems. The first one, especially important in the case of naturally occurring antibodies, is the purity of the antibody preparation used for the assay, since even a trace enzyme contaminant may mimic a high antibody turnover number (8). The second problem is the development of sensitive, continuous, and accurate methods for detection and monitoring of such low activity (9, 10). Here we describe a reproducible method of purification of DNA-hydrolyzing autoantibodies from human sera. We also demonstrate the advantages of the flow linear dichroism (FLD) technique for quantitative and qualitative characterization of the interaction of these catalytic antibodies with supercoiled (sc) plasmid DNA. EXPERIMENTAL PROCEDURESChemicals. All chemicals were from Sigma and Merck. Enzymes were obtained from Boehringer Mannheim.Plasmid DNA. Plasmid pUC19 was isolated as described elsewhere (11). More than 95% of the isolated plasmid DNA was in the sc form, judging by 1% agarose gel electrophoresis (AGE). Antibody Isolation. Antibodies in 5 ml of serum were precipitated twice with 50% saturated ammonium sulfate; this was followed by chromatography on a Pharmacia HR 5/5 staphylococcal protein A fast-performance liquid chromatography (FPLC) column, as described in ref. 13 (p. 310).Antibody Purification. Samples isolated with protein A were dialyzed twice for 4 hr against 500 vol of buffer A (20 mM Tris HCl, pH 9.0) at 4°C and applied to a Pharmacia HR 5/5 Mono Q FPLC column...

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Confocal spectral imaging (CSI) technique was used for quantitative analysis of the uptake, subcellular localization, and characteristics of localized binding and retention of anticancer agent mitoxantrone (MITOX) within human K562 erythroleukemia cells. The CSI technique enables identification of the state and interactions of the drug within the living cells. Utilizing this unique property of the method, intracellular distributions were examined for monomeric MITOX in polar environment, MITOX bound with hydrophobic cellular structures, naphthoquinoxaline metabolite, and nucleic acid-related complexes of MITOX. The features revealed were compared for the cells treated with 2 microM or 10 microM of MITOX for 1 h and correlated to the known data on antitumor action of the drug. MITOX was found to exhibit high tendency to self-aggregation within intracellular media. The aggregates are concluded to be a determinant of long-term intracellular retention of the drug and a source of persistent intracellular binding of MITOX. Considerable penetration of MITOX in the hydrophobic cytoskeleton structures as well as growing accumulation of MITOX bound to nucleic acids within the nucleus were found to occur in the cells treated with a high concentration of the drug. These effects may be among the factors stimulating and/or accompanying high-dose mitoxantrone-induced programmed cell death or apoptosis.

Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.