Horseradish peroxidase folding

Bjemeld E.J., Foldes-Papp Z., Kail M., Rigler R., Single-molecule surface-enhanced Raman and fluorescence correlation spectroscopy of horseradish peroxidase, J. Phys. Chem. B 2002 106 1213-1218. 

The rate constants of kn and kx obtained using Eq. (24) reveal that (i) the activity of Fem-TAMLs in bleaching Safranine O (k ) increases more than 10-fold when the tail ethyl groups of la are replaced by fluorine atoms in lk. The rate constant kn for lk equals l(rM 1s 1 at 25°C, a value that corresponds to those found for the reactivity of horseradish peroxidase Compound II... 

Tyramide signal amplification This procedure, designated as a catalyzed reporter deposition (CARD) or tyramide signal amplification (TSA), takes advantage of horseradish peroxidase (HRP) from an HRP-labeled secondary antibody to catalyze in the presence of hydrogen peroxide the oxidation of the phenol moiety of labeled tyramine. On oxidation by HRP, activated tyramine molecules rapidly bind covalently to electron-rich amino acids of proteins immediately surrounding the site of the immunoreaction. This allows an increase in the detection of an antigenic site up to 100-fold compared with the conventional indirect method with no loss in resolution. 

The rates of asymmetric sulfoxidation of thioanisole in nearly anhydrous (99.7%) isopropyl alcohol and methanol catalyzed by horseradish peroxidase (HRP) were determined to be tens to hundreds of times faster than in water under otherwise identical conditions (Dai, 2000). Similar effects were observed with other hemo-proteins. This dramatic activation is due to a much higher substrate solubility in organic solvents than in water and occurs even though the intrinsic reactivity of HRP in isopropyl alcohol and in methanol is hundreds of times lower than in water. In addition, the rates of spontaneous oxidation of the model prochiral substrate thioanisole in several organic solvents was observed to be some 100- to 1000-fold slower than in water. This renders peroxidase-catalyzed asymmetric sulf-oxidations synthetically attractive. 

Smith, A. T., Santama, N., Dacey, S., Edwards, M., Bray, R. C., Thomeley, R. N. F., and Burke, J. F., 1990, Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca and heme, J. Biol. Chem. 

Figure 6 (a) The five redox states of horseradish peroxidase (FIRP) as determined from the high-resolution crystal strucmre of horseradish peroxidase (HRP). (b) Diagram showing the overall fold and active site environment containing key residues in catalysis of horseradish peroxidase (HRP)... 

The reconstitution process was adopted for enhancing ET of peroxidase and for establishing direct electrical communication of horseradish peroxidase with electrodes. Apo-horseradish peroxidase (apo-HRP) was reconstituted with a dyad consisting of ferrocene tethered to hemin, (14), Fig. 3-14. The resulting reconstituted HRP revealed enhanced ET features and the oxidation of 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, by HO was ca. 2.5-fold faster in the presence of the (14)-reconstituted HRP as compared to the native HRP. 

Add lOOpL of horseradish peroxidase/anti-M13 antibody conjugate (diluted 5,000-fold in PBT buffer). 

Is lipid-assisted folding a widespread phenomenon and possibly applicable to soluble proteins The erythrocyte membrane contains about 20-mole % of PE that is almost exclusively localized in the inner leaflet and is in contact with highly concentrated heme-containing proteins. The refolding of the denatured soluble and heme-containing enzyme horseradish peroxidase (HRP) was followed in the presence and absence of liposomes made up of different phospholipids (Debnath et al., 2003). Remarkably, dimyristoyl-PE (a bilayer-forming... 

Chemical ionization mass spectrometric detection has been explored for the detection of methyl hydroperoxides However, fluorometry has dominated the current detection schemes for the organic peroxides. Typically, a nonfluorescent substrate is oxidized by the peroxide to generate a fluorescent product. These methods are sufficiently sensitive for accurate measurement of the peroxides in the low ppt by volume. For example, the peroxidase-catalyzed dimerization of p-hydroxyphenylacetic acid (POPHA) occurs in the presence of a peroxy group at elevated pH. The formation of the fluorescent dimer, detected by excitation at 310 nm and emission at 405 nm, is proportional to the concentration of the peroxide. The most common peroxidase catalyst used for this reaction is horseradish peroxidase (HRP). Cost and stability issues with the use of HRP led to the use of other catalysts, such as metalloporphyrins or phthalocyanine complexes. Another fluorescent reaction scheme involves the oxidation of the nonfluorescent thiamine (vitamin Bi) to the fluorescent thiochrome by the peroxide group. This reaction is catalyzed by bovine hematin. This reaction is 25-fold more sensitive for H2O2 than for the organic peroxides. 

Targeted drug delivery systems for the anticancer drug Taxotere were built from jS-CyD by monoconjugation with mannosyldendritic branches. Binding inhibition of horseradish peroxidase-labeled Concanavalin A to yeast mannan by mannosylated yS-CyDs was examined. The IC50 values of 95, 96, 97, 98, and 99 were 800, 780, 91, 110, and 8 pM, respectively. 97 solubilized Taxotere similarly to monobranched CyDs [34]. The solubility of Taxotere in water can by increased 1000-fold by 97. 

E.J. Bjerneld, Z. Foldes-Papp, M. Kail, and R. Rigler, Single-Molecule Surface-Enhanced Raman and Fluorescence Correlation Spectroscopy of Horseradish Peroxidase, J. Phys. Chem. B 106, 1213 (2002)... 

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