Violeta G. Janolino

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A.

Abstract The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10−2 min−1. This provides a t 1 2 of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10−2 min−1 and 7 × 10−3 min−1 for uncatalyzed autoxidation and 4 × 10−2 min−1 and 1.1 × 10−2 min−1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.

A spectrophotometric assay for biotin-binding sites of immobilized avidin

A rapid and sensitive spectrophotometric assay was developed for the measurement of biotin-binding sites of immobilized avidin. The method is based on the reaction of avidin with excess biotin followed by assay of the unbound biotin using the HABA (2-[4′-hydroxyazobenzene] benzoic acid) method. Three solids possessing variable amounts of monomeric avidin were examined; viz., succinamidopropyl-controlled-pore glass (CPG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B**), and crosslinked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB1. Results indicate that the total biotin-binding sites of monomeric avidin immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0.093, and 0.218 µmol biotin per mL beads, respectively. Assays for exchangeable biotinbinding sites gave values greater than 90% of the total sites. The spectrophotometric HABA method described is an alternative to assays based on tracers, thus the handling of radioactive material is avoided.

Sulfhydryl oxidase-catalyzed formation of disulfide bonds in reduced ribonuclease

Sulfhydryl oxidase isolated from bovine skim milk membrane vesicles catalyzes de novo formation of disulfide bonds with the substrates cysteine, cysteine-containing peptides, and reduced proteins using molecular oxygen as the electron acceptor. Initial rates for sulfhydryl oxidase-catalyzed oxidation of reduced ribonuclease exhibited typical Michaelis-Menten kinetics at low substrate concentrations. Substrate inhibition of the oxidative activity was observed at ribonuclease concentrations greater than 40 microM, similar to that observed with reduced glutathione or other small thiol substrates. The inhibition was more pronounced when ribonuclease activity was used to monitor the rates, presumably due to concentration-dependent formation of nonnative disulfide bonds. Thus, a maximum in the rate of regain of ribonuclease activity was observed at a 40 microM concentration, while optimum recovery was observed at 30 microM. The Michaelis constant obtained with reduced ribonuclease is 17.4 microM which corresponds to a sulfhydryl concentration of 0.14 mM, a value that compares favorably with the best small thiol substrate, reduced glutathione. Disulfide-containing intermediates in the oxidation pathway, as determined by ion-exchange chromatography of alkylated reaction mixtures, appeared to be similar for air oxidation and enzyme-catalyzed oxidation of the protein. The pH optimum, tissue location, and kinetic characteristics of sulfhydryl oxidase are compatible with a suggested physiological function of direct catalysis of disulfide bond formation in secretory proteins or indirect participation through provision of oxidized glutathione for protein disulfide-isomerase-catalyzed thiol/disulfide interchange.

A spectrophotometric assay for solid phase primary amino groups

A rapid, sensitive, and convenient spectrophotometric assay was developed for the measurement of amino groups on solid supports. This method is based on the reaction of amino groups of solids with an excess ofo-phthaldialdehyde (OPA) and subsequent quantitative determination of unreacted OPA by reaction with glycine. Four solids possessing variable quantities of amino groups were examined. Results indicate that about 70% of the total surface amino concentration (determined by the microKjeldahl method) are available for ligand attachment. Unlike the spectrophotometric 2,4,6-trinitrobenzenesulfonic acid method, the OPA spectrophotometric assay is more rapid, sensitive, and convenient, and unlike the spectrofluorimetric OPA, it does not require sophisticated instrumentation.

Binding of Lactobacillus reuteri to fibronectin immobilized on glass beads

Human fibronectin was immobilized on glass beads. The beads were used to evaluate binding of Lactobacillus reuteri to fibronectin. Organisms bound to the glass beads were detected using fluorescence microscopy after treatment with acridine orange. This binding was confirmed and quantified with the use of [3H]-labelled organisms. Three strains of Lactobacillus reuteri, three strains of Lactobacillus acidophilus and one strain of Lactobacillus fermentum were tested for binding capacity. L. reuteri strain 1063 exhibited a strong binding to the immobilized fibronectin, and L. acidophilus 1754 showed a slight binding. The binding of L. reuteri to the fibronectin was mediated by a protein as judged by the absence of binding after treatment of the bacteria with proteolytic enzymes. Treatment of the bacteria with urea, SDS and heat (80 degrees C) also reduced binding. Treatment of the bacterial cells prior to the assay with fibronectin interfered with binding. Albumin did not show this interaction.

Structural transitions of soluble and immobilized chymotrypsinogen A in urea and guanidinium chloride as measured by fluorescence.

Abstract A cylindrical flow-through quartz cell was designed for measuring fluorescence changes associated with structural transitions in proteins immobilized by covalent attachment to insoluble matrices. Chymotrypsinogen A was immobilized by covalent attachment to derivatized porous glass beads. Conformational transitions in both native, soluble chymotrypsinogen and glass-bound chymotrypsinogen were assessed from fluorescence emission spectra obtained in 0 to 8 m urea and in 0 to 7 m guanidinium chloride. Evidence for the complete reversibility of such transitions in this zymogen was provided by comparing spectra generated by the native zymogen exposed to a given concentration of denaturant with spectra recorded for a mixture of the native zymogen and completely denatured zymogen at the same final concentration of denaturant. The observed transition appeared to follow a two-state mechanism. First order kinetics of unfolding and of refolding were observed in the transition region of the immobilized protein by monitoring fluorescence changes after rapidly adjusting the concentration of denaturant; apparent first order rate constants at pH 7 and 25 °C averaged 0.016 min −1 . Neither the chemistry of the immobilization reactions nor the microenvironment of the surface appears to affect the stability of the native zymogen or the refolding of denatured chymotrypsinogen. Thus, it appears that immobilization of proteins can provide a means for investigating conformational transitions which, due to such complicating secondary reactions as protein-protein interactions and autolysis, cannot otherwise be examined.

Characteristics of sulfhydryl oxidase isolated by a simple chromatographic procedure

Abstract Permeation chromatography of whey or centrifugally clarified solutions of (NH4)2SO4-whey precipitates on controlled-pore glass (3000 A pore diameter) yielded a substantially purified form of the enzyme which eluted in the void volume. This fraction obtained directly from whey appeared to have the least protein contamination as demonstrated by various types of gel electrophoresis, lack of detectable reactivity with rabbit antibovine xanthine oxidase antibody and high specific activity.

[39] Continuous treatment of ultrahigh-temperature sterilized milk using immobilized sulfhydryl oxidase

Publisher Summary This chapter describes the treatment of ultrahigh-temperature sterilized milk using immobilized sulfhydryl oxidase. Sulfhydryl oxidase (SOX) is an iron-containing, glycomembrane enzyme existing primarily in the membrane vesicle fraction of skim milk. Unlike other enzymes that have been developed for use in immobilized forms, SOX is a membrane enzyme, and consequently, certain considerations are important to the choice of immobilization conditions. Sulfhydryl oxidase may be purified by the solubilization of the membrane vesicles with nonionic detergent and isolation of this enzyme by transient covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. At present, however, the quantities obtainable by this procedure prevent its commercial application. Several methods have been developed for the preparation of active isolates directly from whey that are, therefore, commercially attractive. Whey is obtained from the skim milk upon removal of the caseins by clotting with chymosin (rennin). This reaction occurs at the pH of milk and results from the hydrolysis of a specific peptide bond in K-casein; thus, other proteins are not proteolyzed in this treatment.