INTRODUCTION TO PEROXIDASES, GLUCOSE …shodhganga.inflibnet.ac.in/bitstream/10603/38455/4/chapter 2.pdf · 2.1.4.2 Oxidative coupling reaction ... further reduced by a second substrate

CHAPTER - 2

INTRODUCTION TO

PEROXIDASES, GLUCOSE OXIDASE,

URICASES AND CATALASES

Chapter-2 Peroxidase, Glucose oxidase, Uricase and Catalase

31

SECTION 2.1: PEROXIDASE

2.1.1 Introduction

Peroxidases (donor: H2O2 oxidoreductase, POD; EC 1.11.1.7) are a class of

enzymes extensively distributed among higher plants, animals and microorganisms

[1]. Most of the peroxidases are heme proteins and contain ferriprotoporphyrin IX as

the prosthetic group having the molecular weight ranging from 30,000 to 150,000 Da.

They catalyze the reduction of peroxides (H2O2) but also oxidize a variety of organic

and inorganic compounds. Peroxidases are involved in balancing and controlling the

biosynthesis of plant growth hormone, serving as a blanching indicator due to its

thermal stability under limited heat treatment, and is widely employed in

microanalysis due to its ability to yield chromogenic products at low concentrations

with relatively good stability [2-4].

Peroxidases have acclaimed a prominent position in biotechnology and

associated research areas and they are one of the most extensively studied groups of

enzymes. Commercially available peroxidase is widely employed for removal of

phenols and amines from industrial wastewater, bleaching of industrial dyestuff,

lignin degradation, fuel and chemical production from wood pulp and in various

organic syntheses [5].

2.1.2 Different classes of peroxidase

The term peroxidase represents a group of specific enzymes, such as NADH

peroxidase (EC 1.11.1.1), glutathione peroxidase (EC 1.11.1.9), and iodide

peroxidase (EC 1.11.1.8), as well as a group of nonspecific enzymes that are known

as peroxidases. Two super families of heme peroxidases have been identified [6]; one

isolated from plants, fungi, and bacteria and another from mammals. The

homologous enzymes of the plant super family have been sub-divided into three

classes [7]

• Class I - Peroxidases of prokaryotic origin which are intracellular

• Class II - Secretory fungal peroxidases and

• Class III - Extra cellular plant enzymes secreted into the cell wall or the

surrounding medium.

2.1.3 Structure of horseradish peroxidase (HRP)

All plant peroxidase enzymes share the same general structure, consisting of

ferriprotoporphyrin IX as a prosthetic group and ten α-helices. The class III


32

peroxidases also contain three extra α-helices, besides a few highly conserved amino

acids and four disulfide bridges [8]. Figure 2.1.1 shows the three dimensional

representation of the X-ray crystal structure of HRP isoenzyme C. The heme group

(colored in red) is located between the distal and proximal domains in which each

contains one calcium atom (shown as blue spheres). α-Helical and β-sheet regions of

the enzyme are shown in purple and yellow, respectively.

Figure 2.1.1. Structure of Horseradish Peroxidase (HRP)

2.1.4 Probable reaction mechanisms of catalytic activity of peroxidase

Peroxidase catalyzes the oxidation of a wide variety of substrates, using H2O2

or other peroxides as the primary substrate. Catalysis of peroxidase is associated with

four types of activity namely, Peroxidic, Oxidative, Catalytic and Hydroxylation

reaction [1]. The plant peroxidase protein sequence is characterized by the presence of

highly conserved amino acids, such as two histidine residues interacting with the

heme (distal and proximal histidines) and eight cysteine residues forming disulfide

bridges. The distal histidine is necessary for the catalytic activity. These histidine

residues are present in all known heme-containing peroxidase sequences [9].

2.1.4.1 Peroxidic reaction

It is the reaction involving peroxidase and H2O2 (or any peroxide) resulting in

the formation of oxygen free radicals or hydroxyl radicals. The generated radicals

react with other activated aromatic co-substrates to form the respective products [10].


33

2.1.4.2 Oxidative coupling reaction

It involves trapping of free radical species of one co-substrate to get oxidized

to electrophillic species, which couples with the coupling agents of other co-substrates

[11] forming an intense colored product that could be characterized by spectroscopic

instruments.

2.1.4.3 Catalytic reaction

It provides an alternative reaction pathway and stabilizes the intermediates by

reducing the transition energy of the reaction, thereby increasing the number of

reactant molecules with sufficient energy to reach the activation energy and to form

the product [12].

2.1.4.4 Hydroxylation reaction

Hydroxylations belong to the oxygen transfer reactions introducing the

hydroxyl group (.OH) into organic molecules, primarily via the substitution of

functional groups or hydrogen atoms [13].

In general, as a whole, catalytic cycle of peroxidase involves distinct

intermediate enzyme forms. In the catalytic cycle of heme peroxidases, reaction of the

Fe(III) “resting state” active site with peroxide produces an unstable intermediate

called compound I that contains Fe(IV)=O porphyrin π-cation radical and a cation

radical with the consequent reduction of peroxide to water, wherein a distal Histidine

residue plays an essential role in the initial two electron oxidation of resting state

enzyme. A distal Arg residue assists in this process [14, 15].

Then, compound I oxidizes an electron donor substrate to give compound II

(same oxyferryl structure, but protonated), releasing a free radical. Compound II is

further reduced by a second substrate molecule regenerating the iron (III) state and

producing another free radical [16]. Figure 2.1.2 shows the peroxidative and

hydroxylic cycle of peroxidase.


34

Figure 2.1.2. Peroxidase reaction cycles.

The hydroxylic cycle (represented with orange arrows) can generate ROS such

as .OH and HOO. by two different routes (i, ii) (Figure 2.1.2). The source of e- and H+

(1) can be either auxin or other reducing molecules. The peroxidative cycle

(represented in green) can oxidize various substrates, represented by XH and X. for

the reduced and oxidized forms, respectively. X. has three major fates: auxin

catabolism (*), cell wall component polymerization (**) and NAD(P)H oxidation

(***) via a non-catalytic reaction (2) (Figure 2.1.2). NAD(P)H oxidation produces

superoxide, which is immediately converted either spontaneously or by superoxide

dismutase to H2O2 and O2 (3) (Figure 2.1.2). Hydroxylic and peroxidative cycles can

both regulate the H2O2 level. The O2.- released during the oxidative cycle by NADPH

oxidase can convert peroxidase into compound III, which catalyzes the generation of .OH from H2O2 in the cell wall.


35

2.1.5 Physiological relevance of peroxidase

Peroxidases are involved in a wide range of physiological processes related to

plant growth and development [17]. They play an important role in the plant defense

system mechanism by the oxidation of endogeneous phenolic compounds to quinones,

which are toxic to the invading pathogenic organisms and pests [18]. It can also

oxidize the growth hormone auxin, as well as other substrates producing H2O2 and

hydroxyl radicals, which are involved in oxidative burst and cell elongation. By

generating hydroxyl radicals (.OH), peroxidases play a crucial role in seed protection

as well as in the initial days of germination by reducing pathogenic attack. The

diversity of the reactions catalyzed by plant peroxidases explains the implication of

heme proteins in a broad range of physiological processes, such as auxin metabolism,

lignin and suberin formation, cross linking of cell wall components, defense against

pathogens [9], and HRP isoenzymes including indole-3-acetic acid (IAA) metabolism

[19]. It often contributes to deteriorative changes in flavor, color, texture, and

mouthfeel in raw and processed fruits and vegetables [3].

2.1.6 Application of peroxidase

In analytical applications the enzyme must be present in saturated amounts, to

make sure that the H2O2 produced in the test is stoichiometrically converted into a

colored product [20]. Some of the most important applications of peroxidase which

are being used have been elaborated in Table 2.1.1.

2.1.6.1 Peroxidase in immunoassay

Enzyme immunoassay or ELISA test, is the most common technique used for

labeling an antibody are simple and reliable way of detecting toxins, pathogens,

cancer risk in bladder and many other analytes [21]. ELISA tests have been developed

for screening monoclonal antibodies against mycotoxins [22], cystic fibrosis mutation

in blood [23], human tumour necrosis factor alpha, using biospecific antibodies [24].

2.1.6.2 Peroxidase in medical diagnostic kits

Peroxidase has been used for analytical applications in diagnostic kits for the

quantification of important biomolecules such as uric acid [25], glucose [26],

cholesterol [27], nucleic acids [28] etc by using their respective oxidase enzymes.

2.1.6.3 Peroxidase as bioremediation of waste water

Lignin peroxidase (LiP) and manganese peroxidase (MnP) may be

successfully used for bio-pulping and bio-bleaching in the paper industry. HRP

catalyzes the oxidation of aqueous phenols by H2O2 to produce free radicals that


36

spontaneously interact to form polymers and oligomers of high molecular weight and

water-insoluble precipitates. These products are precipitated from the solution and can

be removed from water or wastewater by filtration or sedimentation [29].

2.1.6.4 Peroxidase as biosensors

Biosensor has attracted considerable attention as the potential successor to a

wide range of analytical techniques owing to its unique characteristic of specificity.

Enzymes immobilized on the electrode surface get oxidized by H2O2 and then reduced

by electrons provided by the electrode. During the electron transfer, electrons act as

second substrate for the enzymatic reaction, resulting in the shift of electrode

potential, with measured current being proportional to the H2O2 concentration [30].

Figure 2.1.3 shows the schematic representation of a biosensor. Peroxidase-based

biosensors have been used for the determination of alcohols, glutamate and choline

[16].

Figure 2.1.3 A schematic representation of a biosensor with electrochemical transducer.

2.1.6.5 Peroxidase in agricultural technologies

Peroxidase has a potential for soil detoxification, for instance, herbicides such

as atrazine and triazine gets bio-transformed to the less toxic compounds by P.

chrysosporium which contains LiP and MnP [31], [32]. The use of peroxidase to

improve dewatering of slimes and for the polymerization of humic acid in soil organic

matter has also been reported. Chlorinated phenols and anilines get transformed in

soil by oxidative and detoxification coupling reactions mediated by laccase,

peroxidase or metal oxides as birnessite [33].

2.1.6.6 Peroxidase in pharmaceuticals

HRP/IAA represents an efficient system for enzyme/prodrug-based anticancer

approach [34]. Another application of plant peroxidases in the field of organic and

polymer synthesis is related to the coupling of catharanthine and vindoline to yield α-

3′,4′-anhydrovinblastine which is a part of most of the curative regimes used in cancer

chemotherapy [35].


37

Table 2.1.1 Peroxidase based analytical methods

Sl.

No.

Analytical Method Reagent co-substrate Analyte determined Reference

01

Spec

trop

hoto

met

er

MBTH-DMAB/HRP/GOD/α-glucosidase H2O2, glucose & maltose [11]

ABTS POD activity-broccoli processing wastes [36]

TMB POD activity in soil [37]

TMPD Inactivation of prostaglandin-H-Synthase [38]

Guaiacol POD activity - plant samples [39]

Benzidine/ p-phenylene diamine POD activity in mitochondrial membrane [40]

o-phenylenediamine (OPD) HRP [41]

Eriochrome Blue Black R (EBBR) Degradation of EBBR, fluorescein [42]

o-dianisidine, TMB & OPD POD mechanism [43]

Alizarin/ H2O2/HCl Alizarin degradation [44]

Phenol/4-AAP O2- and SOD activity in vegetables [45]

MBTH/DBZ H2O2 and glucose [46],

2,4-DMA; PPDD-DMAB; PPDD-NEDA HRP and H2O2 [47-49]

Pyrogallol/Veratryl alcohol Effect of veratryl alcohol on LiP [50]

Catechol, o-dianisidine, OPD POD activity [51]

Phenol-4-sulphonic acid & 4-AAP H2O2 and HRP activity [52]

Pyrocatechol/aniline HRP activity [53]


38

02 ELISA Dopamine Dopamine in serum [54]

03 Chemiluminescence-

CL

Luminol HRP [55]

4-(1,2,4-triazol-1-yl)phenol/luminol H2O2 in rain water [56]

04 Flow injection

analysis

3-Aminophthalic acid/Silane; Phenol/AAP Glucose [57, 58]

Luminol, p-iodophenol Sulfamethoxypyridazine in milk sample [59]

05 GC-MS Allergenic eugenol Eugenol removal from rose essential oil [60]

06 Potentiometric 4-fluorophenol H2O2 [61]

07 Photo electrochemical

immunoassay

CdS quantum dots/photoactive antibody-

antigen

Mouse IgG (antigen, Ag) [62]

08

Am

pero

met

ric

Amine group containing polymers H2O2 quantification [63], [64]

Cyanuric chloride [65]

Halloysite & chitosan nano composite H2O2 in milk samples [66]

Dichlofenthion/Organophosphorus hydrolase Dichlorofenthion, pesticides [67]

Polyacrylamide microgels Acetaminophen [68]

Poly(2,5-DMA) Glucose [69]

09 Electro chemical

immunoassay

p-aminophenol Cucumber mosaic virus [70]

3-Hydroxyl-2-amino pyridine α-Fetoprotein [71]

4-chloro-1-naphthol Cancer biomarker-Prostate antigen [72]

o-tilidine Carcinoembryonic Antigen [73]

10 Microtiter plate reader Al(III)Octaethylporphyrin/4-fluoro phenol Glucose [74]

11 Chronometric Ascorbic acid/Benzidine HRP and tea enzyme [75]


39

12 Resonance scattering

assay

Tetradecyldimethylbenzylammonium

chloride

Glucose [76]

13 Capillary electrophoresis Phenol/4AAP/Amberlite IRA-743 resin H2O2 in honey and minerals [77]

14 LC/MS Bisphenol A (BPA) BPA removal from water, waste water [78]

15

Spec

trof

luor

imet

ry

3-(4-hydroxyphenyl) propionic acid H2O2 and Glucose [79]

Amplex red; Fluorescent gold nanocluster H2O2 [80], [81]

Sesamol (3,4-methylenedioxy phenol) Thyroid stimulating hormone [82]

Tyrosine HRP activity [83]

Dihydroxyphenoxazine HRP and H2O2 [84]

Homovanillic acid H2O2 in honey samples [85]

1-Hydroxypyrene Ozone in the air [86]

Hydroxynaphthaldehyde thiosemicarbazon H2O2/-O-O-H bond in PEG [87]

16 ECIS Polymyxin H2O2 and O2 reduction, biofeul cells [88]

Polyacrylamide/SWCNT H2O2 [89]

(CaCO3–AuNPs) inorganic composite [90]

17 Voltammetry-ELISA Alkaline phosphatase/3-indoxyl phosphate Pneumolysin-toxin respiratory to infection [91]

3,3′-diaminobenzidine CEA [92]

18 Batch process Diethyl aminoethyl cellulose

α-naphthol removal in waste water [93]

19 UV irradiation Semiconducting iron-doped titanate Peroxidase activity of HRP

[94]


40

20

Cyc

lic v

olta

mm

eter

Polyaniline/MWCNT

H2O2

[95]

4-ethylnylphenyl [96]

(3-aminopropyl)trimethoxy silane/1,4-

benzoquinone titanate nano tube

[97]

Au NPs –thionine/chitosan [98]

Poly(3,4-ethylenedioxythiophene) - Poly(

styrene sulfonic acid)/Au nanocomposite

[99]

Bismuth oxide (Bi2O3) NPs/ MWCNT [100]

Anthraquinone 2-carboxylic acid [101]

Hydroquinone/Nanoclay Glyphosate [102]

Poly(diallyldimethylammonium chloride) NO2• radicals induced DNA damage [103]

21 Conductivity cell Polyethylene terephthalate/ABTS HRP in Sub femtoliter volume [104]

22 Coulometric biosensor 1,4-hydroquinone/γ-aminopropyl-

diethoxymethyl-silane

H2O2 molecules present in cells [105]

Note: 1. ECIS: Electrochemical Impedance Spectroscopy; CV: Cyclic Voltammeter; MWCNT: Multi walled carbon nanotubes;

SWCNT: Single walled carbon nanotubes; NPs: Nano particles.

2. ABTS: 2,2-Azinobis(3-ethylbenzthiazoline-6-sulfonicacid; MBTH: 3-methyl-2-benzothiazolinone hydrazone hydrochloride; DMAB:

Dimethylamino benzoic acid; TMB: 3,3′,5,5′- Tetramethylbenzidine; TMPD: tetramethyl-p-phenylenediamine; DBZ: 10,11-dihydro-5H-

benz(b,f)azepine); 2,4-DMA: 2,4-Dimethoxyaniline; PPDD: Paraphenylenediamine dihydrochloride; NEDA: N-(1-naphthyl)

ethylenediamine dihydrochloride; 4-AAP: 4-aminoantipyrine; POD: Peroxidase; HRP: Horseradish peroxidase; CEA: Carcinoembryonic

Antigen; LiP: Lignin peroxidase; PEG: Polyethylene glycol.


41

SECTION 2.2: GLUCOSE OXIDASE

2.2.1 Introduction

Glucose oxidase (GOD) (β-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4)

is a flavin containing glycoprotein with high-mannose type carbohydrate content of

10-16% of its molecular weight. The carbohydrate moieties are either N or O- which

are glycosidically linked to the protein. The enzyme activity was first reported by

Muller (1928) [106] in extracts of Aspergillus niger and subsequently, this enzyme

was purified. Müller in 1928 established that the enzyme catalyzes the oxidation of

glucose to gluconic acid in the presence of dissolved oxygen. The fungal enzyme is a

homo dimer made up of two identical subunits each having molecular weight of

approximately 80,000 Daltons [107]. Each monomer contains a non-covalently bound

FAD molecule, which is acting as a redox carrier in catalysis [108]. Sources of GOD

include extracts from Aspergillus niger, Penicillium amagasakiense, Penicillium

vitale and Penicillium glaucum. Isolation of GOD from a number of sources has been

reported and these include red algae, citrus fruits, insects, bacteria and moulds [109].

GOD is produced industrially as a by-product of the gluconic acid fermentation from

A. niger, P. amagasakiense and P. vitale [110].

GOD is the most widely employed enzyme as an analytical reagent and

especially it is useful for the selective determination of glucose, which is an analyte of

clinical as well as of industrial importance. The relatively low cost and good stability

make the glucose/GOD system, a very convenient model for method development

particularly in the area of biosensors.

2.2.2 Structure of Glucose oxidase

GOD is composed of two polypeptide chains of approximately equal

molecular weight held together by disulfide bonds with carbohydrate content

amounting to 16 %. The primary structure of GOD from Aspergillus niger has single

polypeptide chain of one subunit of 583 amino acid residues [111]. The crystal

structure of the enzyme was solved at 2.3 A° resolutions. The protein is glycosylated,

containing between 11 and 30 % carbohydrates: mostly amino- and neutral-sugars

[112], [113]. Dissociation of subunits is possible only under denaturing conditions

and is accompanied by the loss of coenzyme FAD [114]. The structure of GOD is

shown in Figure 2.2.1.


42

Figure 2.2.1. Structure of Glucose oxidase from Aspergillus Niger

2.2.3 General reaction mechanism of Glucose oxidase

GOD catalyzes the oxidation of β-D-glucose using molecular oxygen as an

electron acceptor to D-glucono-δ-lactone, which subsequently gets hydrolyzed

spontaneously to gluconic acid and H2O2 [115], and the reaction involves two steps

one reductive and another oxidative as shown in Scheme 2.2.1.

In the reductive half reaction, GOD catalyzes the oxidation of β-D-glucose to

D-glucono-δ-lactone, which is non-enzymatically hydrolyzed to gluconic acid.

Subsequently the FAD ring of GOD gets reduced to FADH2 [116]. In the oxidative

half reaction, the reduced GOD is reoxidized by oxygen to yield H2O2. The H2O2 is

cleaved by catalase to produce water and oxygen. Witteveen et al., (1992) found that

the enzyme lactonase (EC 3.1.1.17) in A. niger to be responsible for catalyzing the

hydrolysis of glucono-δ-lactone to gluconic acid [117].

Scheme 2.2.1 General reaction mechanism of GOD in presence of glucose as a

substrate


43

2.2.3.a The probable reaction pathway occurring in the colorimetric method

Enzymatic method of determination of blood glucose level using a

chromogenic substrate involves the following mechanism of generation of the colored

reaction product for spectrophotometric method.

2.2.3.b The probable reaction pathway in the electrochemical method

The electrocatalytic processes that occur at the electrode surface can be

depicted as follows

2.2.4 Physiological relevance of Glucose oxidase

GOD in culture filtrates of Talaromyces flavus is responsible for inhibition of

germination of microsclerotia of V. dahliae, a plant pathogen and it is a major factor

in biocontrol of V. dahliae by T. flavus [118]. The inhibitory effect of GOD activity

on germination and melanin formation secreted by T. flavus, retards hyphal growth

and kills microsclerotia of V. dahliae and Sclerotium rolfsii in vitro, probably by

generating toxic peroxide in soil [119, 120]. It is also involved in the biocontrol of

Verticillium wilt of eggplant by T. flavus [121].

GOD gene expression of T. flavus in cotton and tobacco reduces fungal

infection by generating H2O2, in the presence of glucose, which is toxic to

phytopathogenic fungi responsible for economically important diseases in many crops

[122]. It is also involved in disease resistance conferred by gene expression for the

transgenic potato plants [123]. Use of GOD show that prolonged exposure to

moderate concentrations of H2O2 decreases insulin receptor signaling for cellular

mechanism in 3T3-L1 Adipocytes [124]. It is also used as antibacterial in

combination with antibiotics for ocular pathogens [125].


44

2.2.5 Applications of Glucose oxidase

GOD has gained considerable commercial importance during the last few

years due to its multitude of applications in chemical, pharmaceutical, clinical,

biotechnology and other industries. Some of its important current applications in

industry have been reviewed below.

2.2.5.1 Biofuel cells

Biofuel cells consist of two electrode set modified by biocatalytic enzymes to

specifically oxidize/reduce substrate cells by converting biochemical energy into

electrical energy. One approach towards the design of an implantable, membraneless

and biocompatible biofuel cell consists of catalyzing the oxidation of glucose at the

anode using either GOD or glucose dehydrogenase enzyme. These enzymes are

coupled to the reduction of dioxygen at the cathode by a dioxygen-reducing enzyme

such as laccase, bilirubin oxidase etc [126, 127].

2.2.5.2 GOD based biosensors for glucose assessment

GOD has been employed as the biological recognition component in a wide

variety of biosensors for glucose coupled with peroxidase. Some biosensors use

sensitive fluorescence measurements, monitoring changes in the intrinsic FAD

fluorescence of GOD [109]. Some of the recent GOD based biosensors have been

listed in Table 2.2.1.

2.2.5.3 Food and beverage additive

In order to prolong shelf life of foods and beverages, GOD/CAT is used to

remove glucose during the manufacture of egg powder, preventing browning for use

in baking industry, in bread and croissants [128]. This enzyme system is shown to

control non-enzymatic browning during fruit processing and puree storage. The

addition of GOD leads to an increase in the elastic and viscous moduli of wheat and

rice flour dough [129]. GOD is also used to prevent color and flavor loss as well as to

stabilize color and flavor in beer, fish, tinned foods and soft drinks by removing

oxygen from foods and beverages [130, 131].

2.2.5.4 Textile industry

GOD has application in the textile industry as a method for producing H2O2

for bleaching and immobilization of GOD covalently on alumina and glass supports

results in higher recoveries [132]. The H2O2 produced when tested for bleaching

scoured woven cotton fabric was found comparable to standard bleaching processes.

No stabilizers were needed since the gluconic acid produced itself acted as a


45

stabilizing agent [133]. GOD is used along with POD in dyeing baths for decoloration

process and bleaching of natural fibers in textile industries [134].

2.2.5.5 GOD as antagonist in various medical fields

GOD is reported to have the best antagonistic effect against different food-

borne pathogens such as Salmonella infantis, Clostridium perfringens and others

[135]. It has been used as an ingredient of toothpaste [136], and in food preservation

[137]. GOD has been proposed as an anticancer drug because of the damage caused to

cancerous tissue and cells as a result of H2O2 formation [138]. GOD has also been

used as anti-microbial agents in oral care products [139]. The H2O2 produced by GOD

acts as a bactericide and has the ability to kill Streptococcus mutans which appears to

be enhanced by the fusion of enzyme with heavy chain antibodies [140].

2.2.5.6 Gluconic acid production and application

GOD is also used as a commercial source of gluconic acid, which can be

produced by the hydrolysis of δ-glucono-1,5-lactone, the end product of GOD

catalysis. Gluconic acid has been used as a food additive to act as an acidity regulator,

in sterilization solution or bleaching in food manufacturing and as a salt in chemical

components for medication. It also used as a mild acidulant in metal and leather

industries. It has even been used in the construction industry as an additive to cement

in order to increase its resistance and stability under extreme weather conditions.

Gluconic acid occurs naturally in honey, fruits and wine [141], [142].

2.2.5.7 GOD in wine industry

GOD has potential for use in wine industry to lower the alcohol content of

wine through the removal of some portion of the glucose before converting to alcohol.

An alternative approach was introduced with the concept of treating grape juice from

mature fruits with GOD to reduce the glucose content (up to 50 % of grape sugar) of

the juice, which after fermentation produces wine with reduced alcohol content [143].

GOD was able to inhibit spoilage of wine through its bactericidal effect on acetic acid

bacteria and lactic acid bacteria during the fermentation process which insists lesser

quantity of preservatives are needed to be added to the wine [144].


46

Table 2.2.1. Glucose oxidase based analytical methods

Sl. No. Analytical Method Reagent co-substrate Analyte determined Reference

01 Spectrophotometer

Au NPs/N-methylphenazonium methyl

sulphate/2,6-dichloroindophenol

Efficiency of Au NP for enzymatic activity of

GOD

[145]

Triphenylmethane dyes/Benzoquinone GOD as bioanodes in biofuel cells [146]

Poly(Styrene-glycidylmethacrylate Kinetic parameters for GOD enzyme [147]

Polystyrene/AuNPs/dendritic surfactant Nano/microstructures on activity of GOD [148]

02 Chemometric - Characteristics of wheat bread dough [149]

03 Hronopotentiometric Polyaniline/Glutaraldehyde/Graphite Electrochemical detection of glucose [150]

04 Flow injection

analysis

4-AAP/4-hydroxy benzoate Glucose in honey [151]

Chitosan–ferrocene/ Glutaraldehyde GOD and Gluconobacter oxydans biosensors [152]

05 Titrimetric - GOD [153]

06 Miniature chip-design GOD/bilirubin oxidase/NADPH Directed evolution of GOD/biofuel cells [154]

07

Am

pero

met

ric

Polyaniline/glutaraldehyde Glucose [155]

1, 1′-dimethylferricinium GOD activity of cultivation of A. niger [156]

Polypyrrole Characterization of GOD biosensor [157]

Poly-L-lysine/Nafion Glucose sensor and biofeul cells [158]

Graphene oxide and concanavalin A Activity of GOD [159]

Poly-m-phenylendiamine/Hexakinase In-vitro analysis of ATP [160]

08 Chemiluminescence

Bis(2,4,6-trichlorophenyl) oxalate/8-

anilinonaphthalene-1-sulfonic acid

17α-hydroxy progesterone and congenital

adrenal hyperplasia in neonates

[161]


47

Luminol; Nafion/peroxyoxalate Glucose, uric acid; biosensor [162], [163]

09 Electro-

chemiluminescence

ZnO NPs/GOD/graphene/luminol Carcinoembryonic antigen-cancer biomarkers [164]

Poly(nickel(II)tetrasulfo phthalocyanine Glucose in serum samples [165]

poly(ethylenimine)/Luminol α-1-fetoprotein [166]

10 Chrono-coulometric IrO/Nafion/Ir sol/GOD/Au support Glucose biosensor [167]

11 Spectropolarimeter ZnO nano particles/GOD Photoelectrochemical - glucose biosensor [168]

12 ECIS/FTIR 1-butyl-3-methylimidazolium Glucose bioanode for biofuel cells [169]

13 Microchip &

Microarray technology

GOD/antibody/hexamethyl disiloxane

plasma -polymerized film

Development of protein chips for proteomics

of α-1-fetoprotein and β2-microglobulin

[170]

14

Cyc

lic

volta

mm

eter

Phenanthrenequinone tetrathiafulvalene Glucose/O2 –Biofuel cell [171]

DNA/Fe2+/CAT/Ru(bpy)3+3 in situ DNA damage [172]

N,N-diethylacrylamide/4-vinyl pyridine Electrochemical glucose biosensor [173]

1,1′-bis(4-carboxybenzyl)-4,4′-

bipyridiniumdibromide/TiO2/Viologen

Electrocatalytic activity of GOD for glucose

biosensor

[174]

Poly(allylaminehydrochloride) Glucose biosensor [175]

15 Electrochemical

immunosensor

p-benzoquinone/AuCl4− Detection of mouse IgG (antigens) [176]

Ferrocene/peptide wire/Ag nano rod α-tumor protein biomarker [177]

Colloidal Prussian blue/Au NPs/SPCE CEA/α-fetoprotein/Tumor markers [178]

16 Capillary

electrophoresis

1,4-benzoquinone/β-cyclodextrin Enzymolysis and enzyme inhibition assay [179]

17 Potentiometer GOD/ZnO/Ag wires Glucose micro sensor [180]


48

18 Spectrofluorimeter Polypyrrole/FAD/GOD Stability of GOD [181]

Poly(vinyl alcohol)-pyrene/ GOD Glucose sensing [182]

Eu(III)-tetracycline GOD, H2O2 [183]

19 F- sensing microtiter

plate wells

Al(III)Octaethylporphyrin/

4-fluorophenol

Glucose in beverages [74]

20 Optical biosensor

arrays

silica sol–gel/tris(4,7-diphenyl-

1,10-phenanthroline)-Ru(dpp)3Cl2)

Glucose in beverages and blood samples [184]

21 ELISA/Capillary

immunosensor

PEG/POD-labeled antibody/antigen/

glucose/ascorbic acid/Amplex red

Human IgG/Drug screening and clinical

diagnostic application

[185]

22 Fluidic calorimeter Sb–Bi thin film as thermopile/GOD Glucose sensor [186]

23 Plasmon-resonance

sensors

Poly(dimethylsiloxane)/Au nano

spheres

Study of interaction of GOD with its natural

substrate glucose

[187]

24 Si - microcantilevers GOD Microcantilever /Glucose Glucose sensor [188]

25 Phosphorescence Mn-doped ZnS quantum dots/1-ethyl-3-

(3-dimethylaminopropyl)carbodiimide

/N-hydroxy succinimide

Glucose in real serum samples [189]

26 ENDOR FAD in GOD G-Tensors of the FAD radicals in GOD [190]

Note: ECIS: Electrochemical Impedance Spectroscopy; ENDOR: Electron-Nuclear Double Resonance; SPCE: Screen printed carbon

electrode; bpy = 2,2′-bipyridine; ATP: Adenosine triphosphate FAD: Flavin adenine dinucleotide; PEG: Poly(ethyleneglycol)


49

SECTION 2.3: URICASE

2.3.1 Introduction

Uricase (UOx) (Urate oxidase, EC 1.7.3.3) is the copper-binding peroxisomal

liver enzyme, which catalyzes the oxidation of uric acid (UA), a product of purine

metabolism into a more water-soluble allantoin through a complex reaction

mechanism, and then it is freely excreted by kidneys along with urine. Hydroxyisouric

acid, the immediate product of UOx reaction is unstable and thus yields allantoin,

CO2 and H2O2, through nonenzymatic breakdown. UOx is utilized in biological

systems for the degradation of purines and it is present in both prokaryotes and

eukaryotes. This highly conserved endogenous enzyme is present in mammals but not

in humans [191], plants (soybean, wheat (Triticum aestivum), broad bean (Vicia faba)

[192], fungi (Candida) [193], yeasts and bacteria (Bacillus fastidiosus) [194]. Two

nonsense mutations found in human UOx gene, confirm at the molecular level, that

UOx gene in humans is non-functional. UA is a powerful scavenger of free radicals

and it has been proposed that it protects hominoids from oxidative damage and

prolongs life span [195].

UOx is used in humans for the control of increased serum UA in patients with

acute tumour lysis syndrome after receiving chemotherapy. Rasburicase (SR 29142),

a recombinant UOx expressed by Saccharomyces cerevisiae has been demonstrated to

be superior to allopurinol (uricostatic agent) in the control of UA [196]. However,

only few case reports address the potential role of UOx for treatment of severe

tophaceous gout in patient with end-stage renal disease and observed regression of

gout tophi [197]. The treatment was well tolerated in all patients and produced no

adverse effects [198].

2.3.2 Structure of Uricase

UOx is mainly localized in liver, where it forms a large electron-dense

paracrystalline core in many peroxisomes [199]. The enzyme exists as a tetramer of

identical subunits, each containing a possible type 2 copper-binding site [200]. UOx is

a homotetrameric enzyme containing four identical active sites situated at the

interfaces between its four subunits. UOx from A. flavus is made up of 301 residues

with molecular weight of 33438 Da. It is unique among the oxidases as it does not

require a metal atom or an organic co-factor for catalysis. Sequence analysis of

several organisms has shown that there are 24 amino acids which are conserved, and


50

of these, 15 are involved with the active site. The structure of UOx is depicted in

Figure 2.3.1.

Figure 2.3.1 Structure of Uricase enzyme

2.3.3 General reaction mechanism of Uricase

UOx catalyzes the in vivo oxidation of UA in the presence of O2 to produce

allantoin and CO2 as oxidation products of UA and H2O2 as a reduction product of O2.

Enzymes from bovine liver [201], bacteria and fungi [202] do not contain copper.

Soybean UOx is also devoid of copper, other transition metals, and common redox

cofactors, thus presenting a mechanistically intriguing problem of how triplet oxygen

is activated to react with the singlet urate molecule [203]. Although allantoin is the

ultimate product that is formed from the oxidation of urate, the evidences suggest that

urate is oxidized by the enzyme to a metastable compound which gets decomposed

into allantoin nonenzymatically. The other reaction products are H2O2 and CO2,

which are derived from C6 of urate [204]. However, the NMR data used to support

the assignment of 5-hydroxyisouric acid as the product of UOx reaction are not

sufficient to rule out other structures. Characterization of the reaction product by

using a full complement of specifically 13C-labeled urates augmented by isotope-

labeling studies has been done in H218O. With the aid of specifically labeled urates, it


51

was also possible to delineate the pathway for the conversion of the enzymatic

reaction product into allantoin [203].

The probable reaction pathways of UOx catalyzing UA occurring in some

colorimetric and electrochemical methods are as follows

2.3.3.a Colorimetric method

Enzymatic method of determination of blood UA using a chromogenic

substrate involves the following mechanism of generation of the colored reaction

product for spectrophotometric method

2.3.3.b Electrochemical method

The electrocatalytic processes that occur at the electrode surface can be

depicted as follows

2.3.4 Physiological relevance of Uricase

UOx belongs to purine degradation pathway and it prevents the accumulation

of UA in blood. The absence of UOx expression in human has both advantages and

disadvantages because UA is a potent antioxidant and it helps to reduce the presence

of free radicals in cells, but in high concentration it can accumulate in plasma and

induce high pathologies that may be fatal [205].

There are reports regarding the protection of neurological damage of cells by

the presence of UOx [206]. It is the absence of a functional UOx gene that

predisposes humans to hyperuricemia and gout. However, rather than being

advantageous, mutational loss of UOx causes fatal UA nephropathy in mouse [207].

Lack of UOx in humans results in plasma UA concentrations that are much higher

than in most mammals. When these concentrations exceed the solubility limit of about

7 mg/dL at physiological pH, UA may nucleate to form crystals in tissues and joints

leading to acute inflammatory response with acute pain [208].


52

Legumes: UOx is also an essential enzyme in the ureide pathway, where

nitrogen fixation occurs in the root nodules of legumes and is converted into

metabolites that are transported from the roots throughout the plant for amino acid

biosynthesis. In legumes, two forms of UOx are found: in roots, the tetrameric form;

and in the uninfected cells of root nodules, a monomeric form, which plays an

important role in nitrogen-fixation [209]. Increase in UOx activity in the presence of

thioredoxin appears to implicate a novel role for thioredoxin in the regulation of

enzyme activities involved in nodule development and nitrogen fixation [210].

Yeast UOx in plant hoppers: Microorganisms such as bacteria and yeasts

catalyze the oxidation of UA produced in purine breakdown to allantoin and

successively to allantoic acid, urea and ammonia. In symbionts UOx plays a key role

in the host’s utilization of stored UA which is essential for the host to grow normally

[211]. In addition, UOx activity was also detected in symbiotic insects, in brown plant

hoppers (Nilaparvata lugens) and the isolated symbionts, but not in aposymbiotic

insects [212].

2.3.5 Applications of Uricase

UOx has gained considerable commercial importance during the last few years

due to its multitude of applications in the chemical, medical, clinical chemistry,

biotechnology pharmaceutical and other industries. UOx has been the subject of much

research mainly due to its recent therapeutic and diagnostic applications.

2.3.5.1 PEG-uricase in the management of treatment-resistant gout and

hyperuricemia

UOx is formulated as a protein drug (rasburicase) for the treatment of acute

hyperuricemia in patients receiving chemotherapy. A PEGylated form of UOx

(poly(ethylene glycol) conjugates) is under clinical development for treatment of

chronic hyperuricemia in patients with "treatment-failure gout". UOx can be useful in

lowering side effect caused by chemotherapy which results from hyperuricemia

treatment as a consequence of tumour lysis [213]. UOx from A. flavus has been used

therapeutically for treating patients with hyperuricemia, including those treated with

cytoreductive drugs for a malignant hemopathy [214].

2.3.5.2 Uricase as biosensors

A brief introduction on principles of biosensors is provided under Section 2.1

of peroxidase chapter, sub section 2.1.6.4. Using the activity of UOx along with other

oxidoreductase enzymes usually peroxidase, many types of biosensors have been


53

developed and reported for the quantification of either UOx activity or H2O2/UA.

Some of the Uricase based methods reported recently in the literature have been listed

in Table 2.3.1.

2.3.5.3 Uricase in medical & clinical analysis

UOx has been used as a drug for rapid lowering of urate level which is needed

potentially in organ transplant patients with gout, tumor lysis syndrome, tophaceous

gout, renal failure, and acute gout attacks [215]. It has been used for the symptoms of

Lesch-Nyhan syndrome and confirmed the possible protection of neurological cells by

UOx enzyme [206]. It is also used as a peroxisomal marker [216] and is potentially a

good system for studying protein sorting into peroxisomes [200]. A recombinant

preparation of purified fungal (A. flavus) UOx (Uricozyme; Sanofi-Synthelabo)

enzyme is now available in the United States for this indication [217].

2.3.5.4 Uricase as polymer-conjugated therapeutic agent for immunological

studies

Polymer conjugation has so far been successfully used to enhance the

therapeutic potential of many pharmacologically active proteins and peptides since it

allows for the alteration of their physicochemical and biological properties improving

permanence in circulation, stability, solubility and reducing immunogenicity [218].

Poly(N-vinylpyrrolidone), poly(N-acryloilmorpholine) linear and branched PEG2, can

improve the immunogenic character of UOx. Immunological studies showed that

antigenicity and immunogenicity of UOx were altered by polymer conjugation to such

an extent that depended upon the polymer composition [213].

2.3.5.5 Uricase for color development in hair-dyeing and fur-dyeing

In usual hair-dyeing practice, the coloring reaction is initiated by mixing H2O2

directly with the organic reactants and such direct use of H2O2 at relatively high

concentrations may damage hair and skin due to the strong oxidizing power of H2O2.

The UOx-induced hair coloring is effective and much milder than the direct oxidation

method using H2O2. UOx can function as a catalyst for p-phenylenediamine oxidation

as well as the H2O2 supplying source in the presence of UA to generate p-

benzoquinonediimine which is used for the preparation of hair-dyes (oxidative

polymerization of monomeric precursors) [219].


54

Table 2.3.1 Uricase based analytical methods

Sl.

No.

Analytical Method Reagent co-substrate Analyte determined Reference

01 Sp

ectr

opho

tom

eter

DHBS/4-AAP Uric acid (UA) in serum and urine [220]

Polyethyleneterephthalate/DHBS/4-AAP Serum urate [221]

Uricase/Catalase Uric acid [222]

Polyacrylonitrile/o-dianisidine/POD/UOx

/polyaniline/K2Cr2O7

UA in serum [223]

4-AAP/p-hydroxy)benzoicacid Serum UA [224]

N-methyl-N-(4-aminophenyl)-3-methoxy

aniline/TOOS/Ascorbate oxidase

UA in human serum [224]

p-hydroxybenzoate/4-AAP Serum UA assay [225]

Tetrazolium salt/CAT/FADH/1-methoxy-

5-methylphenazinium methyl sulfate

UA in serum [226]

Tribromophenol-4-AAP-POD UA in serum [227]

02 Molecular absorption

diode array

spectrometer

UOx-autotransducer molecular absorption

properties of HRP

UA in synthetic serum samples [228]

03 Chemometric tris(1,10-phenanthroline) /ferritin

Fe(phen)3]3+

AA, UA and DA in serum and

urine

[229]


55

04 Flow injection analysis POD/ferrocene/CPE/Nafion/UOx/GOD H2O2 & D-glucose & UA in serum [230]

Poly(N,N-dimethylaniline) Detection of UA & Ascorbic acid [231]

05 Capillary

electrophoresis

Luminol/K3[Fe(CN)6] UA in urine and serum samples [232]

06 Chemiluminescence Pentacene/Peroxalate NPs/ UOx/alginate H2O2 and UA biosensor [233]

Microfluidic paper/Rhodanine UA biosensor [234]

07 Chemiluminescent

Biosensor

Diethylaminoethyl/Imidodiacetic acid/

GOD/GCE/luminol/Urate

Choline, glucose, glutamate,

lactate, lysine and UA

[235]

08 Infrared reflection

absorption spectroscopy

UOx onto Langmuir monolayers of stearic

acid/ Langmuir–Blodgett (LB) films

UA in Blood [236]

09 Bipotentiostatic Dodecylsulfate/poly(N-methylpyrrole) Detection of uric acid [237]

10

Am

pero

met

ric

Au NPs/MWCNT/carbodiimide linkage UA in serum [238]

Polystyrene/Polymaleimidostyrene UA biosensor [239]

Polyaniline UA in serum & real samples [240],

[241]

Amino acid nano composites UA biosensor [242]

Chitosan-poly(thiophene-3-boronic acid) UA in serum [243]

Gelatin/Glutaraldehyde/Teflon Urinary UA [244]

2-(2-mercaptoethylpyrazine)/4,4′-

dithiodibutyric acid

UA biosensor [245]


56

11 Chronoamperometry SPCE/Cobalt phthalocyanine/cellulose

acetate/Polycarbonate membrane

Uric acid in urine [246]

3-aminopropyltriethoxysilane/Bis[sulfo

succinimidyl]suberate/indium-tin-oxide

UA biosensor [247]

12

Spectrofluorimeter UOx/Uric acid/Glycine/OH− buffer Uric acid in blood serum samples [248]

o-Phenylenediamine Serum UA assay [249]

Pyronine Y/Cu(II)/H2O2 UA in Urine [250]

Polyurethane-UOx-UA/Ru(dpp)3TMS2 UA in human blood serum [251]

Amplex red/silca sol-gel Uric acid in urine, serum & blood [252]

13 CV Poly(o-aminophenol) Uric acid in serum [253]

Theonine/SWCTs Endogenous & Physiological UA [254]

NiO/Titanium/Glass substrate UA biosensor [255]

Thionine-SWNTs UA in Cell lysate-serum samples [254]

Mercaptobenzimidazole/Ascorbic acid UA in human serum [256]

14 Coulometric Porous carbon felt electrode Uric acid in urine [257]

15 Conductometric Polyaniline-poly(n-butylmethacrylate)/Poly

(vinylmethylether)-poly(vinylethyl ether)

Urea and UA detection using

Urease and UOx

[258]

16 Reagentless biosensor UOx/ZnS Quantum Dots/L-cys biosensor UA biosensor [259]

17 96-well microtiter plate 8-azaxanthine, competitive inhibitor-UOx Urate oxidase activity rasburicase [260]

18 HPLC Cimetidine UA & creatinine in urine [261]

Semen samples diluted in Dithiothreitol/ AA and UA in human seminal [262]


57

Ethanol-NaH2PO4 used as a mobile phase plasma

19 LC-MS m/z 167.0, corresponds to urate anion, and

m/z 169.0, corresponds to 1,3-15N2-UA

anion (isotope labeled UA as an internal

standard)/Trichloroacetic acid

Intracellular (Human umbilical

vein endothelial cells)/

Extracellular (plasma & urine) UA

[263]

20 Potentiometric assay ZnO/Au/Nafion UA in human blood serum [264]

21 Photo array sensor Metal oxide semiconductor/polymeric

enzyme biochip/UOx-POD

Serum UA [265]

22 Oxygen sensor UOx/Eggshell membrane/O2 electrode/UA UA in serum & urine [266]

23 Calorimetric biosensors UOx-Oxalate oxidase/biothermochips Kidney calculus indices [267]

24 Aqualytic O2 meter UOx-epoxy resin/polyamine cross linker Serum UA [268]

25 pH sensor Poly-N-isopropylacrylamide/Eu2Ti2O7 UA biosensor [269]

Sm2TiO5/Si substrate/UOx- alginate film Serum UA [270]

Note: CV: Cyclic voltammeter; DHBS: 3,5-Dichloro-2-hydroxybenzenesulfonic acid; 4-AAP: 4-aminophenazone; UOx: Uricase; UA:

Uric acid; FADH: Formaldehyde dehydrogenase; AA: Ascorbic acid; DA: Dopamine; CPE: Carbon paste electrode; GCE: Glassy carbon

electrode; SPCE: Screen printed carbon electrode; LC/MS- Liquid Chromatography-Tandem Mass spectrometry; TOOS: N-ethyl-N-

(hydroxy-3-sulfopropyl)-toluidine.


58

SECTION 2.4: CATALASE

2.4.1 Introduction

Catalase (H2O2 oxidoreductase, EC 1.11.1.6, CAT) comprises of four

ferriprotophorphyrin as prosthetic groups per molecule containing proteins that

include a variety of cytochromes, globins and peroxidases and is an important part of

body's antioxidant defenses, and is present in almost all living organisms. Acetobacter

peroxidans and Shigella dysenteriae [271] are exceptions and usually strict anaerobes

lack this enzyme. CAT is the typical marker enzyme of peroxisomes [272] and

participates in the degradation of H2O2 generated in the acyl-CoA oxidase reaction.

CAT defends against oxidative stress associated with pathologic conditions arising

out of cancer, diabetes, atherosclerosis, reperfusion injury, neurodegenerative disease

and aging [273].

In human tissues CAT plays a central role in controlling H2O2 concentration

by converting it into O2 and H2O [274], otherwise known as “catalatic” reaction. The

liver, erythrocytes and kidney are rich in CAT and more than 98 % of blood CAT is

derived from erythrocytes [275]. Other tissues such as brain, pancreas and serum have

CAT in very low concentration [276]. The normal range of CAT concentration in

healthy subjects is 120 U/mL ± 20 U/mL, whereas its concentration may vary

depending upon the condition of patients [277]. CAT, along with superoxide

dismutase (SOD) and glutathione peroxidase (GSH-Px), scavenges most of the levels

of O2-derived free radicals in mammalian cells and they function together as a

somatic oxidant defense [278]. Because ROS potently damages tissues, their final

conversion by SOD and CAT to harmless molecular O2 and H2O represents a

powerful antioxidative system, for preventing oxidative modifications of DNA,

proteins and lipids by blocking the chain reactions of free radicals produced in the

human body [279]. Free radicals are also responsible for many previously

unexplained diseases such as rheumatoid arthritis, Alzheimer’s, hyper-tension,

myocardial ishchemia, liver cells injury and carcinogenesis [280]. Hence,

measurement of CAT activity under certain conditions is valuable to assess the status

of vital defense system.

2.4.2 Structure of Catalase

Catalase is a tetramer of four polypeptide chains, each having over 500 amino

acids. It contains four porphyrin heme groups that allow the enzyme to react with

H2O2. The optimum pH of human CAT is approximately 7, and has a fairly broad


59

maximum. The pH optimum of other Catalases varies between 4 and 11 depending on

the species [281]. The optimum temperature also varies by species. The structure of

Catalase is shown in Figure 2.4.1.

Figure 2.4.1 Structure of Catalase (CAT)

2.4.3 General reaction pathway of CAT and its native iron states

HRP and CAT are the two enzymes which catalyze the decomposition of same

substrate: H2O2. However, the mechanisms of decomposition of H2O2 by these two

enzymes are completely different. HRP reduces H2O2 to H2O and CAT catalyzes the

breakdown of H2O2 into H2O and O2. In a ‘catalatic’ reaction of CAT enzyme, H2O2

oxidizes the heme iron of the resting enzyme to form an oxyferryl group with a π-

cationic porphyrin radical, termed compound I (Cpd I) as shown in Equation (1).

Where ‘Enz’ is the resting or ground state of the enzyme and Cpd refers to other states

of CAT.

The enzyme is first oxidized to a high-valent iron intermediate, known as Cpd

I which, in contrast to other hydroperoxidases, is reduced back to the resting state by

further reacting with H2O2. It is found that the Cpd I-H2O2 complex evolves to a Cpd

II-like species through the transfer of a hydrogen atom from the peroxide to oxoferryl

unit. The complete reaction sequence may involve two mechanisms that may be


60

operative: a His-mediated mechanism [282], which involves the distal His as an acid-

base catalyst mediating the transfer of a proton (associated with an electron transfer),

and a direct mechanism, in which one hydrogen atom transfer occurs [279]. Isotope

labeling kinetic studies on CAT have demonstrated that both the oxygen atoms of the

O2 molecule originate from the same H2O2 molecule [283]. Based on the crystal

structure of CAT, Fita and Rossmann (1985) proposed that the two hydrogens of

H2O2 are sequentially transferred to the oxoferryl unit of Cpd I, with the distal His

playing an active role in the reaction. Reaction 2 is a two-electron redox process and

clarifying whether it actually involves a two-electron transfer elementary step would

pose CAT in clear contrast with peroxidases, for which the resting state is restored in

two one-electron reduction processes [284].

Another state of CAT, Cpd II, results from the reduction of Cpd I by a single

electron as in Equation (3)

Recently, Rovira [285], provided evidence for the formation of a

hydroxyferryl form of Cpd II as in Equation (4)

Cpd II is formed during the catalatic reaction. It is inactive in the catalatic

reaction, but reverts spontaneously and relatively slowly to the active form (Enz).

Thus, during exposure to H2O2 that is generated at a constant rate, CAT can reach a

steady state in which much of it is inactive.

Mammalian CAT also has limited ability to act as a peroxidase. An example

of such a ‘peroxidatic’ reaction is the formation of acetaldehyde from ethanol and

H2O2. After the initiation of Cpd I in Equation (1), the reaction that takes place is

shown in Equation (5)

Peroxidatic activity is relatively slow. But in contrast, the catalatic rate of

mammalian CAT is highest among the known enzymatic rates. The catalatic rate is

simply proportional to H2O2 concentration over a wide range of H2O2 and it does not

follow the Michaelis–Menten kinetics [286]. Moreover, efforts made to determine the

reaction rates at high H2O2 concentrations are thwarted by the inactivation of CAT

that occurs at such levels. As a consequence, peroxidatic reactions are noticeable at


61

low H2O2 concentrations, but catalatic reaction predominates at higher H2O2

concentrations [287].

2.4.4 Physiological relevance of Catalase

CAT protects hemoglobin by removing more than half of the H2O2 generated

in normal human erythrocytes, which are exposed to substantial O2 concentrations

[288]. CAT activity has been observed in human placenta during early gestation

period [289]. Presence of L-DOPA, dopamine, hydroquinone and other

autooxidizable compounds in the fetal cells of rat inhibit the CAT activity resulting in

cell damage under mesencephalic cultures [290]. Studies on lymphocytes which is

activated by H2O2, stimulated in vitro by ROS to induce angiogenesis showed that

only enzyme CAT could block the activation [291]. Progesterone and various

synthetic steroids with progestin potencies counteract cell growth induced by H2O2,

through potent induction of CAT activities, in breast cancer cells and normal human

epithelial breast cells [292]. Increase in CAT activity has been observed in HIV

infected AIDS patients. Increases in serum CAT activity correlated with increase in

serum H2O2 scavenging ability and reached levels which decreased exogenous H2O2-

mediated injury to in vitro cultured endothelial cells without altering neutrophil

bactericidal activity or mononuclear cell cytotoxicity [293]. The toxic effects of ROS

are neutralized in the lens of eye by antioxidants such as ascorbic acid, vitamin E,

glutathione system (GSH peroxidase and reductase), SOD and CAT [294] and also in

the cataractous lenses of the type 2 diabetic and in senile group [295].

The decrease of CAT activity in malignant phenotype of mouse keratinocytes

studied u in-vitro model results in tumor progression suggesting that CAT should be

present in a sufficient amount to reduce the advancement of tumor growth [296].

Presence of GSH-Px, glutathione-S-transferase, CAT, Cu–Zn SOD activities and

malondialdehyde levels in erythrocytes of patients with non-small-cell lung cancer

(NSCLC) and small-cell lung cancer (SCLC), indicate significant changes in

antioxidant defense system in NSCLC and SCLC patients, which may lead to

enhanced action of oxygen radical [297].

A study of plasma thiobarbutyric acid-reacting substances (TBARS), blood

GSH (reduced glutathione) concentrations and erythrocyte antioxidant enzyme

activities (SOD/CAT/GSH-Px) in regularly menstruating women with ovulatory and

anovulatory menstrual cycles has made possible for the classification of subjects as

either ovulating or non-ovulating [298].


62

CAT has been implicated as an important factor in inflammation, mutagenesis,

prevention of apoptosis and stimulation of a wide spectrum of tumors [279]. It was

Fridovich [299], who suggested in 1975 that O2- and H2O2 interact to form the highly-

reactive hydroxyl radical according to the following equation

The reaction of oxidase systems like Hypoxanthine oxidase/xanthine results in

the formation of O2- which involves in the degradation breakage of single-stranded

DNA molecules and attacks on sugar moiety and the SOD and CAT enzymes protect

the DNA from these effects in presence of iron salts. Xanthine oxidase/xanthine

systems also render degradation and loss of viscosity in human synovial fluid system

wherein SOD and CAT act as antioxidants by protecting the biological system [300].

CAT has been reported for the prevention of target cells like nucleated cells,

which are readily killed by an enzymatically generated flux of superoxide. Addition

of human and murine erythrocytes blocks lethal damage to target cells and also

protects heterologous somatic cells against exogenous oxidant challenge [278]. Both

human SOD and CAT have been used in vivo as antioxidant therapy scavenging a

very small fraction of total oxidant production resulting decrease in lipid peroxidation

[301].

2.4.5 Applications of Catalase

The ability to degrade H2O2 has made CAT as one of the most industrially

significant enzymes. Immobilized CAT has useful applications in various industrial

fields including the removal of H2O2 used as oxidizing, bleaching or sterilizing agent

[302] and in the analytical field as a component of H2O2 or glucose biosensor

systems. Some important applications of Catalase are the following.

2.4.5.1 Catalase as biosensors

A brief introduction on principles of biosensors is provided in the second

chapter of Section 2.1: Peroxidase chapter, sub section 2.1.6.4. Using the catalatic

activity of CAT along with other oxidoreductase enzymes many types of biosensors

have been developed for the quantification of CAT activity, H2O2, and glucose. Some

of the recent Catalase based biosensors have been listed in Table 2.4.1.

2.4.5.2 Catalase in analysis of pollutants/pesticides in biological samples

Water pollutants such as pesticides, insecticides and heavy metals are the main

substances that were detected by amperometric biosensors via an enzyme alteration


63

process [303]. CAT is used for the determination of nitrite [304], cyanide [305], 3-

amino-1,2,4-triazole [306], [307] and azide [308] based on their inhibition effect on

enzyme activity in water and fruit juice samples.

2.4.5.3 Catalase in medical or clinical analysis

CAT, SOD and polyHb (poly hemoglobin) and a biodegradable polymeric

nano encapsule of Hb and enzymes (SOD-CAT, as lipid membrane artificial red

blood cells) have been used as blood substitutes [309]. PolyHb–SOD–CAT stabilize

the cross linked Hb resulting in decreased oxidative iron and heme release and also

reduce the formation of methemoglobin during the preparation of polyhemoglobin

[310]. Encapsulated CAT has been used successfully as an antioxidant against the

toxic effects of H2O2 in acatalesemic mice [308]. The first generation polyHb blood

substitutes (phase III clinical trials) have shown important clinical potential for certain

clinical conditions especially for short-term use in surgery [311].

CAT has also been used as a sperm motility extender in the cryopreservation

of bovine semen. Bovine sperm motility in egg yolk tris glycerol extender has been

protected by the use of pyruvate, metal chelators, bovine liver or oviductal fluid CAT

[312]. CAT can be used as a therapeutic agent in a variety of human diseases as CAT

can inhibit ROS-mediated tissue injury and tumor metastasis [313]. PEGylation-CAT

can effectively prevent the increase in the expression of epidermal growth factor

receptor metastatic tumor growth by detoxifying ROS as well as the peroxidation

[314]. CAT was encapsulated in biocompatible flexible non-ionic sugar esters (SEs)

nano-vesicles for topical administration in wound healing [315].

2.4.5.4 Catalase in food industry

In food industry, CAT has been used in the disposal of H2O2 prior to cheese

making [316]. CAT of cetyltrimethylammonium bromide-permeabilized cells was

effective in removing residual H2O2 from H2O2-treated fresh milk/heat pasteurized

milk [317]. In dairy industry, when H2O2 is used as a germicide for cold

pasteurization CAT is employed to remove the residual H2O2 present [318]. Other

important applications of CAT include desugaring of egg white [319], stabilization of

beverages by oxygen removal [320] and enzymic production of gluconic acid and

gluconic acid–fructose mixture from invert sugar [321].


64

Table 2.4.1 Catalase based analytical methods

Sl.No Analytical Method Reagent co-substrate Analyte determined Reference

01 S

pect

roph

otom

eter

Trinder’s reagent/Uric acid-Triton X-100 CAT in plasma, erythrocytes and liver [273]

Sodium cyanide CN contamination in aquatic biota [305]

Ammonium molybdate Serum CAT activity [322]

Ironporphyrin/3-amino-l,2,4-triazole CAT-hemolysates human, rat, mouse blood [323]

GOD/Aldehyde dehydrogenase CAT activity in erythrocytes [324]

K2Cr2O7/acetic acid CAT activity [325]

4-AAP/phenol/peroxidase CAT activity in soils [326]

p-nitrophenol p-nitrophenol inhibitory effect on CAT [327]

4-amino-3-hydrazino-5-mercapto-1,2,4-triazole CAT activity in small tissue samples [328]

o-Dianisidine CAT activity in honey samples [329]

3,3'-Diaminobenzidine-Glutaraldehyde Cytochemical detection of CAT [330]

NADP-NADPH Serum CAT activity [331]

NADPH-CAT Protection of CAT by NADPH [332]

Tetrahydrobiopterin (THB)-Ascorbate THB effect on ascorbate CAT [333]

Choline oxidase/4-aminopent-3-en-2-one Determination of Choline in liquor [334]

Oxalateoxidase/Aldehyde dehydrogenase Oxalate in serum and Urine samples [335]

Pyrocatechol-Isoniazid CAT activity-mycelia mats/culture media [336]

Methylhydroperoxide/Amino-1,2,4-triazole Formation of com I of CAT is investigated [337]


65

02 FIA L-phenylalanine/L-amino acid oxidase L-phenylalanine in serum samples [338]

CNBr/Xanthine oxidase/Glyceraldehyde Monitoring inactivation-CAT by glycation [339]

03 Plate reader Ferrous ions/Thiocyanate Neural cell cultures of mesencephalon [340]

04 Amperometric ([Zn2CrABTS]LDH)/([Zn3AlCl]LDH)/NO2- Detection of nitrite in water samples [341]

AuNPs/MWCNTs/Chitosan CAT activity in liver homogenates of rats [342]

Polyacrylamide gel/Pt/Ag wire H2O2 biosensor [343]

05 Batch technique Glutaraldehyde/Chitosan/Cu(II) Metal sorption and CAT immobilization [344]

06 Calorimeter Uricase-CAT-tris buffer pH 9.0 Measurement of serum uric acid [345]

07 Chemiluminescence Luminol/Hypochlorite (NaOCl)/NaN3 Human erythrocytes and rat hepatocytes [346]

Dopamine/Luminol Study of auto oxidation of DA [347]

08 Conductometric HRP-CAT/Nitrite Detection of nitrite in water samples [304]

09

Cyc

lic v

olta

mm

eter

AuNPs/Graphene-NH2/GCE H2O2 biosensor [348]

Polyelectrolyte-encapsulated CAT/Au H2O2 biosensor [349]

MWCNTs/GCE H2O2 reagentless biosensor [350]

NiO/GCE H2O2 & nitrite reduction biosensor [351]

Cysteine/Si sol–gel/GCE/Au/Al3+ Neurotransmitter detection of CAT & Al3+ [352]

SWCNTs/Chitosan/GCE H2O2 biosensor and nitrite detection [353]

SOD-CAT/ Hg electrode Antioxidant properties of SOD and CAT [354]

10 Disk flotation

method

CAT CAT activity measurement [355]

11 ESR technique Sodium azide-CAT-H2O2 Detection of formation of Azidyl radical [356]


66

12 O2 Microsensor CAT/H2O2/degassed borate buffer CAT activity in green coffee sample [357]

13 Gasometric method H2O2/CAT Erythrocyte CAT activity [358]

14 HPLC Glutathione/o-phthalaldehyde derivative CAT activity in lysed human erythrocytes [359]

15 IMAC cryogel CibacronBlue/Poly(acrylamide-allyl glycidyl

ether) cryogel was chelated with Fe3+ ions

Effect of pH, protein conc., flow rate, and

temp. on ionic strength on CAT activity

[360]

16 ECIS 1-butyl-3-methylimidazoliumhexafluoro

phosphate/MWCNTs/GCE

Detection of ultra traces of H2O2 [361]

17 Oxygen sensor GOD-CAT/Pt-gas permeable membrane Glucose detection in blood [362]

18 pH sensitive

hydrogel

Sulfadimethoxine/GOD-CAT Glucose responsive hydrogel [363]

19 Batch plug-flow type Glutaraldehyde/6-aminohexanoicacid florisil Characterization and application [364]

20 Polarography Triphenylphosphine oxide SOD/CAT activity-human red blood cells [365]

21 Potentiometric HRP-CAT/H2O2/4-fluoro phenol CAT positive microorganisms [366]

22 Pyrolysis 2,2'-Azo-bis-(2-amidinopropane) (ABAP) Study of deactivation of SOD and CAT [367]

23 Radiometer Polypropylene/Nephrophan membrane H2O2 biosensor [368]

24 Raman spectra Isoniazid (INH)-CAT-peroxidase Activity of antituberculosis antibiotic INH [369]

25 Spectrofluorimeter Europium (III)–tetracycline Catalase activity [370]

26 Thermostat Gelatin/(CAT-Ca2+)/Glutaraldehyde Calcium in milk and water samples [371]

27 Titrimetric method KMnO4 CAT activity in waste water [372]

Note: 4-AAP: 4-aminoantipyrine; GCE: Glassy Carbon Electrode; Luminol: (5-amino-2,3-dihydro-1,4-phthalazinedione; IMAC:

Immobilized metal ion affinity chromatography cryogel; ECIS: Electrochemical Impedance Spectroscopy.


67

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INTRODUCTION TO PEROXIDASES, GLUCOSE …shodhganga.inflibnet.ac.in/bitstream/10603/38455/4/chapter 2.pdf · 2.1.4.2 Oxidative coupling reaction ... further reduced by a second substrate