Spectrophotometric quantification of horseradish peroxidase with o-phenylenediamine (original) (raw)
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Study of the active site of horseradish peroxidase isoenzymes A and C by luminescence
Biochemistry, 1985
Luminescent derivatives of horseradish peroxidase isoenzymes A and C were prepared by replacing the heme with protoporphyrin IX or mesoporphyrin IX. The isoenzymes showed about the same binding affinity as the active enzymes for hydroxamic acid derivatives. The fluorescence and phosphorescence yields and lifetimes of the porphyrin derivatives at room temperature decreased in the presence of substrates. Under site-selection conditions (low temperature and narrow-band excitation), resolution in the emission spectra of the porphyrin derivatives could be obtained, proving that the spectra are inhomogeneously broadened. Addition of substrate resulted in a change in distribution of the 0-0 lines in the resolved spectra. The results are discussed in terms of a distribution of sites which are altered by substrate. H o r s e r a d i s h peroxidases (HRP) * are heme glycoproteins prosthetic group but with different physicochemical and kinetic which use hydrogen peroxide to oxidize a wide variety of compounds (Yamazaki & Yokota, 1973). Horseradish contains at least seven isoenzymes, all with protoheme IX as the This work was supported by National Science Foundation Grant PCM 84-0844 and Swedish Medical Research Council Grants 03X-6522 and 7130. properties (Shannon et al., 1966). A basic (C) and an acidic (A) isoenzyme together account for about 75% of the total amount. HRP C has higher enzyme activity and tighter ' Abbreviations: HRP, horseradish peroxidase; HRP A, horseradish isoperoxidase type A2 (acidic); HRP C, horseradish isoperoxidase type C2 (basic); NHA, 2-naphthohydroxamic acid; PP, protoporphyrin IX; MP, mesoporphyrin IX.
Talanta, 1997
Peroxidase entrapment in different Sol-Gel matrices was successful. The enzyme did not show a decrease in activity for at least 2 months as well as storage at room temperature and dry condition for periods exceeding 3 weeks. It was evident that the enzymatic activity was a function in the type of the alkoxysilane precursor. In addition, the optimum temperature which resulted in maximum enzymatic activity was also dependent on the type of Sol-Gel matrix. Excellent results were obtained for the determination of glucose in serum samples using soluble glucose oxidase in conjunction with the Sol-Gel entrapped peroxidase. The enzymatically produced hydrogen peroxide is oxidized by the entrapped peroxidase yielding oxygen which oxidizes the faint blue variamine blue into the intensely violet colored species (the molar absorptivity is about 1.8 x 104 1 mol-~ cm-~). The characteristics of this chromogenic system as well as optimized conditions for its use in the spectrophotometric determination of enzymatically generated hydrogen peroxide were investigated. Excellent agreement between the results obtained by the proposed method and the widely used standard method, utilizing a commercial reagents kit, was always observed. © 1997 Elsevier Science B.V.
Clinical Chemistry and Laboratory Medicine, 1989
Enzyme immunoassays frequently incorporate the use of horseradish perosidase äs the enzyme label. This enzyme usually catalyses the oxidation of a ehromogen which can be quantified after termination of the enzyme reaction. A chromogen widely used for this purpose is S^'^S'-tetramethylbenzidine. The two electron oxidation of tetramethylbenzidine yields a component with an absorbance maximum at 450 nm. If the enzyme reaction is terminated by lowering of the pH (< 1.0), an additional increase of the absorbance at 450 nm is observed. It is shown that this additional increase is partly due to a l .4-fold increase in the molar lineic absorbance of oxidized tetramethylbenzidine, caused by the acidic pH, äs well äs a quantitative shift of the existing equilibrium between tetramethylbenzidine, oxidized tetramethylbenzidine and their charge-transfer complex. The total absorbance increase upon acidification of the reaction mixture depends therefore on the reaction conditions äs well äs the reaction coordinate.
Kinetics of the reaction of compound III of horseradish peroxidase with hydrogen peroxide and NADPH
The kinetic study of the reaction of Compound III of horseradish peroxidase with reduced nicotinamide adenine dinucleotide phosphate (NADPH) was investigated in phosphate buffer as a function of ionic strength at 25 °C. Two successive reactions involving increase and decrease in absorbance with time were observed. Each reaction was first order with respect to the concentration of horseradish peroxidase. The observed rate constants were ionic strength dependent within the range of 0.06 – 0.30 M. The logarithmic values of the rate constants against the square root of the ionic strength showed that both NADPH and Compound III of horseradish peroxidase are of same ionic charges in the first reaction but different ionic charges in the second reaction.
Journal of Inorganic Biochemistry, 2002
It is generally assumed that the putative compound I (cpd I) in cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I of peroxidases and catalases and many synthetic cpd I analogues. In these systems one oxidation equivalent resides on the Fe(IV)=O unit (d(4), S=1) and one is located on the porphyrin (S'=1/2), constituting a magnetically coupled ferryl iron-oxo porphyrin pi-cation radical system. However, this laboratory has recently reported detection of a ferryl iron (S=1) and a tyrosyl radical (S'=1/2), via Mössbauer and EPR studies of 8 ms-reaction intermediates of substrate-free P450cam from Pseudomonas putida, prepared by a freeze-quench method using peroxyacetic acid as the oxidizing agent [Schünemann et al., FEBS Lett. 479 (2000) 149]. In the present study we show that under the same reaction conditions, but in the presence of the substrate camphor, only trace amounts of the tyrosine radical are formed and no Fe(IV) is detectable. We conclude that camphor restricts the access of the heme pocket by peroxyacetic acid. This conclusion is supported by the additional finding that binding of camphor and metyrapone inhibit heme bleaching at room temperature and longer reaction times, forming only trace amounts of 5-hydroxy-camphor, the hydroxylation product of camphor, during peroxyacetic acid oxidation. As a control we performed freeze-quench experiments with chloroperoxidase from Caldariomyces fumago using peroxyacetic acid under the identical conditions used for the substrate-free P450cam oxidations. We were able to confirm earlier findings [Rutter et al., Biochemistry 23 (1984) 6809], that an antiferromagnetically coupled Fe(IV)=O porphyrin pi-cation radical system is formed. We conclude that CPO and P450 behave differently when reacting with peracids during an 8-ms reaction time. In P450cam the formation of Fe(IV) is accompanied by the formation of a tyrosine radical, whereas in CPO Fe(IV) formation is accompanied by the formation of a porphyrin radical.
Detection of Enzymatically Generated Hydrogen Peroxide by Metal-Based Fluorescent Probe
Analytical Chemistry, 2011
b S Supporting Information H ydrogen peroxide (H 2 O 2 ) is generated as a result of substrate oxidations by some oxidoreductases. 1 For example, glucose oxidase catalyzes the conversion of D-glucose to gluconolactone, which is accompanied by the generation of H 2 O 2 . By quantifying the amount of H 2 O 2 , interesting biological molecules such as specific enzymes can be indirectly quantified. A combination of N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) and horseradish peroxidase (HRP) is frequently used in assays of enzymes that produce H 2 O 2 as well as their substrates such as D-glucose, uric acid, and acetylcholine. 2,3 This method mainly relies on two characteristics of peroxidases: (1) specific and fast reaction with H 2 O 2 and (2) subsequent generation of metal-based oxidants. H 2 O 2 -activated HRP rapidly converts nonfluorescent Amplex Red to highly fluorescent resorufin (j F = 0.75 4 ). However, Amplex Red cannot be oxidized by H 2 O 2 itself. On the other hand, there are other types of fluorescent H 2 O 2 probes that do not require the aid of peroxidases. In general, these fluorescent probes are small organic molecules and are converted from a weakly or nonfluorescent form to a strongly fluorescent form through the oxidation of probes by H 2 O 2 itself. 2,5À15 One exception is the EuÀtetracycline complex that exhibits strong luminescence when the water ligand is replaced with H 2 O 2 without the occurrence of a redox reaction. 16 Wolfbeis et al. successfully applied the Eu-based H 2 O 2 probe to detect enzymatically produced H 2 O 2 . 16,17 Recently, Chang and co-workers succeeded in developing a new class of fluorescent probes triggered by the oxidative conversion of phenyl boronic esters to phenol derivatives by H 2 O 2 . 8 It has been shown that boronate-based fluorescent probes respond to H 2 O 2 with high selectivity, but the response is rather slow (t 1/2 ∼ 30 min). Very recently, Nagano et al. reported a fluorescent probe having benzil as a reactive moiety for H 2 O 2 ; the reactivity of the fluorescent probe is comparable to that of boronate-based probes. Herein, we present the synthesis and properties of a metalbased fluorescent probe for H 2 O 2 called MBFh1, which has an iron complex as a reaction site for H 2 O 2 and a 3,7-dihydroxyphenoxazine derivative as the fluorescent reporter unit. Scheme 1 shows the synthesis of MBFh1 and our strategy for H 2 O 2 detection, wherein the iron complex reacts quickly with H 2 O 2 to form oxidants and then the oxidants convert the closely appended nonfluorescent 3,7-dihydroxyphenoxazine moiety to resorufin in an intramolecular fashion.