Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.     

Phosphorus 31 solid state NMR characterization of oligonucleotides covalently bound to a solid support.

31P cross polarization (CP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra were acquired for various linear and branched di- and tri-nucleotides attached to a controlled pore glass (CPG) solid support. The technique readily distinguishes the oxidation state of the phosphorus atom (phosphate versus phosphate), the presence or absence of a protecting group attached directly to phosphorus (cyanoethyl), and other large changes in the phosphorus chemistry (phosphate versus phosphorothioate). However, differences in configurational details remote from the phosphorus atom, such as the attachment position of the ribose sugar (2'5' versus 3'5'), or the particulars of the nucleotide bases (adenine versus uridine versus thymine), could not be resolved. When different stages of the oligonucleotide synthetic cycle were examined, 31P CPMAS NMR revealed that the cyanoethyl protecting group is removed during the course of chain assembly.     

DNA methyltransferases: Activity minigel analysis and determination with DNA covalently bound to a solid matrix

We describe two methods that facilitate detection and characterization of DNA methyltransferases: activity gel analysis and the use of DNA-cellulose or DNA-Sepharose in DNA methylation reactions. The first permits identification of catalytic subunits, determination of the influence of proteolysis, and evolutionary or developmental studies. The second allows accurate and fast determination of DNA methyltransferase activities in crude extracts and during purification.     

Properties of penicillin amidase covalently bound to cellulose matrices]

Properties of penicillinamidase (PA) covalently bound with the cellulose matrix were studied. The efficiency of the binding depended on the bind type and purity of the native enzyme taken for binding. Stability of the immobilized PA (IPA) was studied at wide pH ranges. The effect of the ion strength, substrate concentration and purity of the native PA on stability of IPA was also investigated. The maximum stability of the enzyme was observed at pH 6.5-7.0 Stability of IPA depended on the purity of the native enzyme. When PA of the diazotized ether of cellulose containing amino groups was used, the enzyme was destabilized. IPA prepared on chlortriazinylcellulose was more stable than the respective native PA almost by I order.     

Air-reoxidation of reduced, denatured chymotrypsinogen A covalently attached to a solid matrix

Bovine chymotrypsinogen A was covalently attached to porous succinylglass beads via the zymogen's amino groups. The bound zymogen was completely reduced in 8 M urea, then allowed to reoxidize, and activated to chymotrypsin. Comparison of the k_o (catalytic coefficient) of this preparation with that of a similar preparation which had never been exposed to reductant showed a 53% recovery of esterolytic activity towards N-benzoyl-L-tyrosine ethyl ester. Values of K_m and K_o for non-reduced, matrix-bound zymogen, following its activation to chymotrypsin, were 12.8 × 10⁻⁵ M and 11.0 sec⁻¹, respectively. Corresponding values for reduced, air-reoxidized preparations were 6.8 × 10⁻⁵ M and 3.1 sec⁻¹.     

Biotinylated antibodies bound to streptavidin beads: a versatile solid matrix for immunoassays.

Streptavidin was covalently bound to commercially available polyacrylamide beads (3-10 microns diameter) by peptide bond formation between the carboxyl groups on the solid matrix and the amino groups of the soluble protein. Biotinylated antibody or lectin was linked to the polyacrylamide beads via the streptavidin molecules. Immunoassays for human IgA1, IgA2, IgE, and vitronectin were developed utilizing the antibody or lectin as a capture ligand. The protein being assayed was quantitated colorimetrically at 492 nm via horseradish peroxidase-conjugated antibodies.     

d-Galacturonandigalacturonohydrolase covalently bound on to a polyacrylamide-type support

d -Galacturonandigalacturonohydrolase was immobilized by covalent coupling on to a polyacrylamide-type carrier BIO Gel CM100, activated by water-soluble carbodiimide. Catalytic properties, stability and action pattern of the immobilized enzyme are reported.     

Localization of pyridoxal-5-phosphate, covalently bound with human hemoglobin. Spectrofluorimetric studies]

Human apohemoglobin tryptophan residues were localized in the regions of the protein globule with restricted mobility. By the method of dynamic quenching of phosphopyridoxyl chromophore fluorescence, the heterogeneity of pyridoxal-5-phosphate molecules covalently bound to the human hemoglobin molecules was determined from the accessibility to solvent. The first four pyridoxal-5-phosphate molecules are localized in the hydrophobic regions of the hemoglobin molecule; at the same time, they have a high mobility. One of these molecules is situated at the site inaccessible to the solvent, which coincides with the anion-binding center of the oxyhemoglobin molecule. The next pyridoxal-5-phosphate molecules modify the surface amino groups of the protein. In the apohemoglobin molecule, the pyridoxal-5-phosphate binding sites are more exposed to the solvent, as compared to hemoglobin. In the hemoglobin molecule modified by pyridoxal-5-phosphate, an effective electron excitation energy transfer from tryptophan residues to phosphopyridoxyl chromophores occurs. The effective distances between tryptophanyls of single subunits of hemoglobin and the covalently bound pyridoxal-5-phosphate molecule were estimated to be 19 A for the alpha-subunit and 17 A for the beta-subunit.     

Properties of alpha-chymotrypsin and cis-cinnamoylchymotrypsin in free, covalently bound to a carrier and microcapsulated states]

alpha-Chymotrypsin and its light-sensitive derivative, cis-cinnamoylchymotrypsin, free and immobilysed (by means of microcapsulation, covalent binding with sepharose 4B and p-aminobenzyl cellulose) are studied. Effect of pH on the hydrolysis rate of N-alpha-acetyl-L-tyrosine ethyl ester by different immobilized enzyme preparations, their acylation with isomeric mixture of cis- and trans-cinnamoylimidazole, reactivation of stable cis-derivatives of different enzyme samples and thermal stability of native and microcapsulated enzyme preparations are investigated.     

Studies on acetylcholinesterase and cholinesterase covalently bound to polymaleinic anhydride.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), respectively, were covalently attached to a cross-linked copolymerisate of maleinic anhydride and butanediol-divinylether. Based on the coupling procedure reported by Brümmer et al. (Brummer, W., Hennrich, N., Klockow, M., Lang, H. and Orth, H.D. (1972) Eur. J. Biochem. 2k, 129--135), a simple method is described which requires only 24 h for completion and provides a sufficient yield. Although a polyanionic carrier was used the Km and k2 values as well as the substrate and pH optima of the bound acetylcholinesterase and bound cholinesterase did not differ considerably from the corresponding values of the free enzymes. Bound acetylcholinesterase and to some extent also bound cholinesterase did not lose any enzymatic activity after storage in saline at 4 degrees C for 140 days.     

A protein covalently bound to ColE1 DNA

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColE1 DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane-treated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColE1 DNA via a phosphodiester bond between dC and the serine residue.     

Adsorption of mutagens to cotton bearing covalently bound trisulfo-copper-phthalocyanine

A method for separating nonpolar mutagens from their dilute aqueous solutions is described. It utilizes the affinity of the mutagens to a phthalocyanine derivative attached to cotton through a covalent bond. For mutagens having 3 or more fused aromatic rings in their structures, efficient adsorption took place on soaking the cotton in their solutions. The mutagens adsorbed can be recovered by elution with ammoniacal methanol. Mutagenicity in smoker's urine, cooked beef, and river water was detected by use of this method.     

Crystal structure of trioxacarcin A covalently bound to DNA

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule ‘caught in the act’ after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 Å resolution by MAD phasing employing brominated oligonucleotides. The DNA–drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3′-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.     

Identification and characterization of a protein covalently bound to DNA of minute virus of mice.

We identified a protein which is covalently linked to a fraction of the DNA synthesized in cells infected with minute virus of mice. This protein is specifically bound to the 5' terminus of the extended terminal conformers of the minute virus of mice replicative-form DNA species and of a variable fraction of single-stranded viral DNA. The chemical stability of the protein-DNA linkage is characteristic of a phosphodiester bond between a tyrosine residue in the protein and the 5' end of the DNA. The terminal protein (TP) bound on all DNA forms has a relative molecular weight of 60,000; it is also seen free in extracts from infected cells. Immunologic comparison of the TP with the other known viral proteins suggests that the TP is not related to the capsid proteins or NS-1.