Catalytically Active Azoaldolase. Preparation on Solid Support

Abstract

A procedure for the coupling at pH 7.2 of p‐carboxy benzene diazonium chloride with rabbit muscle aldolase supported on phosphocellulose is described and some of the spectroscopic, structural and catalytic features of the material obtained are reported. The tetrameric azoenzyme is homogeneous in disc gel electrophoresis even in the presence of 8 M urea. Twelve molecules of the reactant are bound to the protein. Eight azocysteines are identified by both spectroscopic studies and amino acid analysis. The presence of one azohistidine is suggested by the spectroscopic data along with the presence of other, as yet unknown, chromophores. The azoaldolase shows unchanged catalytic properties using both D‐fructose 1,6‐bisphosphate and d‐fructose 1‐phosphate as substrates, as compared with the native enzyme. The pH profile of the enzyme activity is broadened towards the alkaline region but no changes occur in the physiological range of pH.

Preparation and characterization of sulfanilazo and arsanilazo proteins

“…The spectra overlay (Figure 5), leaving little doubt that sulfhydryls are modified in azo-YADH. Very similar spectra have been reported for the reaction of aldolase with diazotized paminobenzoic acid (Felicioli et al, 1975) and sulfanilic acid (Burton & Waley, 1967). Since the molar absorptivity of the model compound is less than 1500 at pH 7 and 380 nm, less than 700 at pH 7 and 410 nm, and 0 at pH 11 and 540 nm, azo coupling of sulfhydryls, like free amines, will not interfere with spectral analysis.…”

“…Analytical Procedures. Sulfhydryls were determined with DTNB (Ellman, 1959) and primary amines with TNBS (Fields, 1972). Concentrations of azo proteins were determined by either the method described under Spectral Analysis or the methods of Bradford (1976) or Lowry (1951).…”

A selective reaction of fructose bisphosphate aldolase with fluorescein isothiocyanate in chicken muscle extracts

“…Furthermore, reaction with an affinity label was shown to prevent aldolase from binding to PC implicating the nucleotide-binding site to be associated with the active site of the enzyme (Palczewski et al, 1985;Palczewski and Kochman, 1987). (Aldolase binds to PC at its active site (Felicioli et al, 1975).) The sites of modification by the affinity labels are within reasonable proximity to the apparent 5 0 -FITC reactive peptide on aldolase.…”