ABSTRACT
Hydrochloric acid hydrolysis of azo proteins in which lysine residues are azo coupled, results in conversion of modified lysines to alpha-amino-epsilon-hydroxy caproic acid plus alpha-amino-epsilon-chloro caproic acid. The latter can interfere with the determination of tyrosine by amino acid analysis. This potential problem can be avoided either by making basic and then neutralizing HCl hydrolysates or by hydrolyzing the protein in methane sulfonic acid.
Subject(s)
Amino Acids/analysis , Azo Compounds/analysis , Lysine/analysis , Norleucine/analogs & derivatives , Proteins/analysis , Chemical Phenomena , Chemistry , Hydrolysis , Ribonuclease, PancreaticSubject(s)
Cobalt , Myosins , Phenanthrolines , Adenosine Triphosphatases/metabolism , Animals , Chymotrypsin , Cobalt/pharmacology , Edetic Acid/pharmacology , Hydrogen-Ion Concentration , Kinetics , Muscles/enzymology , Myosins/metabolism , Peptide Fragments , Phenanthrolines/pharmacology , RabbitsABSTRACT
This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.
Subject(s)
Carboxypeptidases , Cobalt , Amino Acids/analysis , Arsanilic Acid/analogs & derivatives , Arsanilic Acid/pharmacology , Carboxypeptidases/metabolism , Circular Dichroism , Cobalt/pharmacology , Hydrogen-Ion Concentration , Kinetics , Peptide Fragments/analysis , Protein Binding , Protein Conformation , Spectrophotometry , Tyrosine/analogs & derivatives , Tyrosine/pharmacology , p-AzobenzenearsonateSubject(s)
Amino Acids , Cobalt , Chelating Agents , Chemical Phenomena , Chemistry , Crystallography , Deuterium , SpectrophotometrySubject(s)
Amino Acids , Acetates , Amino Acids/chemical synthesis , Chelating Agents , Chemical Phenomena , Chemistry , Circular Dichroism , Copper , Heterocyclic Compounds , Ligands , Nickel , ZincSubject(s)
Amino Acids/chemical synthesis , Cobalt , Deuterium , Isotope Labeling , Chelating Agents , Methods , Optical Rotation , StereoisomerismABSTRACT
As analogs for cobalt(III) complexes of bidentate azotyrosine in proteins, several cobalt(III) complexes of bidentate azophenols have been prepared and characterized. Tris(2-phenylazo-p-cresolato)cobalt(III) was shown to be a poor model complex for this purpose; while cobalt(III) complexes of either 2-phenylazo-p-cresol or 2-(4-carboxyphenylazo)-4,5-dimethylphenol with the linear tetradentate ligand ethylenediamine-N,N'-diacetic acid are very useful as model complexes. The complexes were characterized by magnetic susceptibility, visible spectra and 1H NMR. In the case of one of the mixed ligand complexes, a detailed description of the stereochemistry was possible. Comparisons of the spectral characteristics of metal complexes with bidentate and tridentate azophenols are made. These model studies are essential to a program involving the site specific modification of proteins with substitution inert metal ions.
