ABSTRACT
The DNA binding of BMS 181176, an antitumor antibiotic derivative of rebeccamycin was characterized by DNA unwinding assays, as well as by fluorescence emission and polarization spectroscopic techniques. Unwinding and rewinding of supercoiled DNA was interpreted in terms of intercalation of BMS 181176 into DNA. BMS 181176 shows an enhanced fluorescence emission upon binding to the AT sequence and no enhancement upon binding to the GC sequence. BMS 181176 appears to be a weaker binder to poly(dAdT).poly(dAdT) compared to doxorubicin and ethidium bromide. When bound to DNA, the rotational motion of BMS 181176 is substantially decreased as evident from the increase in fluorescence polarization. BMS 181176 exhibits a range of binding strengths depending on the DNA. This is demonstrated by the Acridine Orange displacement assay using fluorescence polarization.
Subject(s)
Antibiotics, Antineoplastic/metabolism , Carbazoles/metabolism , DNA/metabolism , Fluorescence Polarization , Glucosides/metabolism , Indoles , Intercalating Agents/metabolism , Acridine Orange/metabolism , Animals , Binding, Competitive , Cattle , DNA/chemistry , DNA, Bacterial/metabolism , DNA, Superhelical/metabolism , Doxorubicin/metabolism , Ethidium/metabolism , Male , Micrococcus/chemistry , Nucleic Acid Conformation , Plasmids/chemistry , Poly dA-dT/metabolism , Salmon , Spermatozoa/chemistry , Thymus Gland/chemistryABSTRACT
The bioaffinity of receptor-ligand interactions is investigated by determining the binding constant (association constant or dissociation constant) of the resulting complex utilizing affinity capillary electrophoresis (ACE). The ACE binding assay was established with a potent immunosuppressant, deoxyspergualin (DSG), that binds specifically to Hsc70, a constitutive or cognate member of heat shock protein 70 (Hsp70) family. Quantitative determination of binding constants under different running buffer systems provide comparative results. The association constants for the interaction between Hsc70 protein and DSG were found to be 5.7 x 10(4) M-1 in a buffer with pH 6.95 and 6.3 x 10(4) M-1 in a buffer with pH 5.30 (or corresponding dissociation constants, 18 and 16 microM, respectively) based on Scatchard analyses. Binding of DSG to a synthetic peptide, SINPDEAVAYGAAVQAAILSGDK, one of the DSG-binding fragments found from tryptic digest of Hsc70 protein, provides further detailed information for the understanding of Hsc70 binding domain. The applicability of using coated capillaries was also evaluated for probing Hsc70-DSG interaction.
Subject(s)
Carrier Proteins/metabolism , Electrophoresis/methods , HSP70 Heat-Shock Proteins/metabolism , Amino Acid Sequence , Binding Sites , Buffers , Capillary Action , Guanidines/metabolism , HSC70 Heat-Shock Proteins , Hydrogen-Ion Concentration , Immunosuppressive Agents/metabolism , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Trypsin/metabolismABSTRACT
An in-house modified microcolumn liquid chromatography (LC) system has been coupled to a PE-SCIEX API III triple-quadrupole mass spectrometer through an ionspray interface for the structural characterization of model glycoproteins, bovine ribonuclease B and human alpha 1-acid glycoprotein. In conjunction with enzymatic digestion approaches using trypsin and peptide-N-glycosidase F, the feasibility of packed-capillary (250 microns I.D.) LC columns, coupled with ionspray mass spectrometry (MS) in a tandem format, have been assessed for glycopeptide mapping and structural determination. This configuration demonstrates a highly promising approach for the determination of glycosylation sites and the corresponding sequence structures of related tryptic fragments. A glycosylated tetrapeptide, Asn-Leu-Thr-Lys with carbohydrate moieties on Asn-34, was readily located for bovine ribonuclease B. Preliminary results using micro-LC-MS also show the identification of a class A carbohydrate attachment on a tryptic fragment of human alpha 1-acid glycoprotein. The microheterogeneity of carbohydrate moieties can be quickly screened using this approach for either tryptic digests or the intact glycoprotein. These methods demonstrate potential applications for structural characterization of recombinant glycoproteins of pharmaceutical interest.
Subject(s)
Chromatography, Liquid/methods , Glycoproteins/chemistry , Mass Spectrometry/methods , Amino Acid Sequence , Animals , Carbohydrate Sequence , Cattle , Humans , Molecular Sequence Data , Spectrophotometry, UltravioletABSTRACT
The esperamicins are members of a class of potent antitumor antibiotics that contain stained diacetylenic ring systems capable of forming DNA-cleaving diradicals upon reaction with thiols. Here we show that the diacetylenic ring core itself determines the sequence specificity for scission of duplex DNA): esperamicin A1, and three products of hydrolysis of the glycon, esperamicins C, D, and E, are found to retain a common sequence preference. The sugar residues exert a strong influence on the cleavage efficiency, presumably by interacting nonspecifically with DNA. The presence of a branch in the DNA is found locally to inhibit scission by esperamicins, and this effect is shown to be due to the core also.
Subject(s)
Aminoglycosides , Anti-Bacterial Agents/metabolism , Antibiotics, Antineoplastic/metabolism , DNA/metabolism , Anti-Bacterial Agents/chemistry , Base Sequence , Carbohydrate Sequence , Electrophoresis, Polyacrylamide Gel , Enediynes , Molecular Sequence DataABSTRACT
A dynamical model for the structure of the human immunodeficiency virus 1 (HIV-1) protease dimer in aqueous solution has been developed on the basis of molecular dynamics simulation. The model provides an accurate account of the crystal geometry and also a prediction of the structural reorganization expected to occur in the protein in aqueous solution compared to the crystalline environment. Analysis of the results by means of dynamical cross-correlation coefficients for atomic displacements indicates that domain-domain communication is present in the protein in the form of a molecular "cantilever" and is likely to be involved in enzyme function at the molecular level. The dynamical structure also suggests information that may ultimately be useful in understanding and further development of specific inhibitors of HIV-1 protease.
Subject(s)
HIV Protease/ultrastructure , Binding Sites , Crystallography , Hydrogen Bonding , Molecular Sequence Data , Motion , Protein Conformation , Water , X-Ray DiffractionABSTRACT
Nitrosation of water-soluble (diethanolamine) and oil-soluble (dodecylmethylamine and dicyclohexylamine) amines in the absence and presence of inhibitors in model anionic and non-ionic emulsions was studied. Nitrosation of diethanolamine occurred at similar rates in non-ionic and anionic emulsions. Surprisingly, dodecylmethylamine and dicyclohexylamine were readily nitrosated in non-ionic emulsions, but not in anionic emulsions. Sodium bisulfite and ascorbyl palmitate were effective inhibitors, but the activity of ascorbic acid was lower. Considerably less effective were potassium sorbate, alpha-tocopherol and butylated hydroxyanisole. The results of this study will help formulators of emulsion products to minimize N-nitrosamine contamination.
