Carbon Tetrachloride (1)

8/10/2019 Carbon Tetrachloride (1)

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Carbon tetrachlorideis theorganic compound with theformula CCl4. It was formerly widely

used infire extinguishers,as a precursor torefrigerants,and as acleaning agent.It is a colorlessliquid with a "sweet" smell that can be detected at low levels. Both carbon tetrachloride and tetra

chloromethane are acceptable names underIUPAC nomenclature.

In the carbon tetrachloridemolecule,fourchlorineatoms are positioned symmetrically as corners

in atetrahedral configuration joined to a centralcarbon atom by singlecovalent bonds.Becauseof this symmetrical geometry, CCl4is non-polar.Methane gas has the same structure, making

carbon tetrachloride ahalomethane.As asolvent,it is well suited to dissolving other non-polarcompounds, fats, and oils. It can also dissolveiodine.It is somewhatvolatile,givingoffvapors with a smell characteristic of other chlorinated solvents.

Uses for carbon tetrachloride

Most carbon tetrachloride is used to make chlorofluorocarbon propellants and refrigerants,though this has been declining steadily. It has also been used as a dry cleaning agent and fire

extinguisher; in making nylons; as a solvent for rubber cement, soaps, insecticides, etc.

How are people exposed to carbon

tetrachloride?Drinking/Eating:People are most often exposed to carbon tet in the environment by

drinking contaminated groundwater. Carbon tet may contaminate groundwater nearlocations where the chemical was improperly disposed. Since the compound is heavy, some

of the spilled liquid will sink through soil and enter groundwater. Carbon tet does not moveeasily with groundwater. Plants do not take up or store carbon tet when they grow incontaminated soil.
http://en.wikipedia.org/wiki/Organic_compoundhttp://en.wikipedia.org/wiki/Chemical_formulahttp://en.wikipedia.org/wiki/Fire_extinguisherhttp://en.wikipedia.org/wiki/Refrigerationhttp://en.wikipedia.org/wiki/Cleaning_agenthttp://en.wikipedia.org/wiki/IUPAC_nomenclaturehttp://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Chlorinehttp://en.wikipedia.org/wiki/Atomhttp://en.wikipedia.org/wiki/Tetrahedronhttp://en.wikipedia.org/wiki/Carbonhttp://en.wikipedia.org/wiki/Covalent_bondhttp://en.wikipedia.org/wiki/Methanehttp://en.wikipedia.org/wiki/Halomethanehttp://en.wikipedia.org/wiki/Solventhttp://en.wikipedia.org/wiki/Iodinehttp://en.wikipedia.org/wiki/Volatility_(chemistry)http://en.wikipedia.org/wiki/Vaporhttp://en.wikipedia.org/wiki/Vaporhttp://en.wikipedia.org/wiki/Volatility_(chemistry)http://en.wikipedia.org/wiki/Iodinehttp://en.wikipedia.org/wiki/Solventhttp://en.wikipedia.org/wiki/Halomethanehttp://en.wikipedia.org/wiki/Methanehttp://en.wikipedia.org/wiki/Covalent_bondhttp://en.wikipedia.org/wiki/Carbonhttp://en.wikipedia.org/wiki/Tetrahedronhttp://en.wikipedia.org/wiki/Atomhttp://en.wikipedia.org/wiki/Chlorinehttp://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/IUPAC_nomenclaturehttp://en.wikipedia.org/wiki/Cleaning_agenthttp://en.wikipedia.org/wiki/Refrigerationhttp://en.wikipedia.org/wiki/Fire_extinguisherhttp://en.wikipedia.org/wiki/Chemical_formulahttp://en.wikipedia.org/wiki/Organic_compound


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Touching:Carbon tet can be absorbed through the skin if a person handles the chemical orcontaminated soil, or bathes in contaminated water.

Breathing:Carbon tet evaporates easily from water. Therefore, a person may be exposedto its vapors when they bathe, cook, or wash with contaminated water.

TOXI EFFE TS

Carbon tetrachloride is readily absorbed after ingestion and inhalation, but more slowlythrough the skin .

Acute exposure to carbon tetrachloride can also cause central nervous system (CNS)

depression as well as gastrointestinal and neurological effects such as nausea, vomiting,abdominal pain, diarrhoea, headache, dizziness, in-coordination, impairment of speech,

confusion, anaesthesia, and fatigue.

The liver and kidney are the major target organs for toxicity following acute inhalation or

ingestion exposure to carbon tetrachloride . Liver damage can occur after 24 hours andin serious cases this can result in painful swollen liver, haemorrages, hepatic coma

and death. Kidney damage with an impairment in function normally occurs 2-3 weeks

after exposure, but in severe cases this can occur within 1-6 days in association with liverfailure.

Acute ocular exposure or skin contact can cause irritation of the eyes and skin. Direct

skin contact with undiluted carbon tetrachloride has been reported to cause a mild burning

sensation with mild redness. Some individuals may be hypersensitive and develop markedswelling, itching and blisters following skin contact.

Chronic inhalation may result in liver and kidney toxicity and neurological effects from

depression of the central nervous system . Neurological and gastrointestinal symptoms

are similar to those for acute exposure, such as depression, nausea and othergastrointestinal effects. In long term repeated dose studies in animals the liver has been

shown to be the most sensitive organ regarding toxicity.

The International Agency for Research on Cancer (IARC) concluded that there is inadequateevidence for the carcinogenicity of carbon tetrachloride in humans. However, based on

evidence in animal studies, IARC has concluded overall that carbon tetrachloride is possibly

carcinogenic to humans (Group 2B). The doses inducing liver tumours in animal studiesare higher than those causing liver cell toxicity, and therefore are considered to arise


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secondary to toxic effects on the liver .

Carbon tetrachloride does not have any significant mutagenic properties.

Data from animal studies indicate that carbon tetrachloride does not have any adverseeffects on development at dose levels below those producing toxicity to the maternal animals.

There are no adequate reproductive toxicity studies on carbon tetrachloride, but

limited data has suggested that exposure to relatively high concentrations of carbontetrachloride may impair fertility.

Health Effects of Acute / Single Exposure

Human Data

General toxicity

Acute exposure to carbon tetrachloride via any route of exposure can cause gastrointestinaland neurological effects in the first 24 hours, such as nausea, vomiting, diarrhoea,

headache, dizziness, depression of conscious level and dyspnoea.The liver and kidney are the major target organs for toxicity following acute inhalation or

ingestion exposure to carbon tetrachloride . Liver damage can occur after 24 hours andin serious cases this can result in painful swollen liver, haemorrage, hepatic coma and death.

Kidney damage with an impairment in function normally occurs 2-3 weeks after

exposure, but in severe cases this can occur within 1-6 days in association with liverfailure.

Adverse effects on the liver can be markedly increased by the co-ingestion of alcohol,

due to hepatioc enzyme induction which results in increased production of toxic metabolites.

Inhalation

Inhalation of carbon tetrachloride may cause rapid depression of the central nervous system,

leading to headache, giddiness, weakness, lethargy and stupor. No effects were reported in

healthy volunteers following exposure to 50 ppm for 70 minutes or 10 ppm for 3 hours. Areview of a number of reports of carbon tetrachloride intoxication led to the conclusion that

no effects were noted up to 80 ppm for 3-4 hours. At higher concentrations nausea,

vomiting, headache, and more severe CNS effects have been noted . Headache anddizziness was also reported following exposure to 250 ppm for 15 minutes.

Carbon tetrachloride is hepatotoxic, the principle features include swollen and tender liver,

elevated serum enzyme levels and jaundice as well as marked liver necrosis with steatosis.

CARBON TETRACHLORIDETOXICOLOGICAL OVERVIEWIngestion

Ingestion of carbon tetrachloride can lead to hepatotoxicity. Single doses of approximately

90-180 mg kg-1 bw caused mild hepatotoxicity (fatty liver) and ingestion of 670 mg/kgresulted in marked hepatotoxicity (severe necrosis) and also nephrotoxicity .Ingestion of carbon tetrachloride can also result in CNS effects, with drowsiness being noted

after ingestion of 300 mg kg-1. No CNS effects were seen at lower concentrations, with only

nausea being reported following ingestion of 100 mg kg-1.


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Dermal / ocular exposure

Carbon tetrachloride can cause irritation of the eyes and skin [4]. Direct skin contact with

undiluted carbon tetrachloride has been reported to produce a mild burning sensation with

mild redness . Some individuals may be hypersensitive and develop marked swelling,itching and blisters following skin contact .

Acute toxicity of carbon tetrachloride is reported to be independent of the route of exposure,therefore dermal exposure to relatively high concentrations may cause similar effectsas for oral and inhalation exposure.

Delayed effects following an acute exposure

Acute exposure to carbon tetrachloride via any route can cause liver damage after 24 hoursor more, renal dysfunction may occur in 1-6 days but may, in many cases, only be apparent

two to three weeks after exposure.

Animal and In-Vitro Data

General toxicity

The liver is the most sensitive target organ following inhalation or ingestion of carbon

tetrachloride in animals. Adverse effects on the kidney, central nervous system and lungsalso occur.

Inhalation

In animals, the hepatic effects of inhalation exposure to carbon tetrachloride are much thesame as in humans, such as elevated serum enzyme levels, steatosis, and centrilobular

necrosis progressing to fibrosis.

Changes in serum enzyme levels indicative of liver damage were seen in rats following 4-

hour exposure to 530 ppm or above. Liver necrosis has been reported following exposure to4800 ppm . Signs of central nervous system depression, such as lack of

coordination, breathing difficulties and unconsciousness have also been observed in animals

exposed to approximately 7000 to 10500 ppm. In another study, rats exposed to


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seen approximately 16 hours after the initial contact .

Application of 0.1 ml into the eyes of rabbits in a Draize test produced mild irritation. The

response was evident at 24, 48 and 72 hours after exposure and recovery was complete

after 14 days .

Health Effects of Chronic / Repeated Exposure

An imal and In-Vitro Data

Inhalation

The liver and the kidneys are the principle target organs following chronic inhalation to

carbon tetrachloride in animals. The predominant signs of hepatotoxicity include steatosis,

elevated serum enzyme levels and centrilobular necrosis .In a subchronic (13 week) study rats and mice were exposed to up to 800 ppm (5192 mg m3)

for 6 hours per day, 5 days per week. In both species microscopic changes were seen in the

liver at autopsy at the lowest dose level (10 ppm; 64 mg/m3). In rats, effects on the blood

were seen at 30 ppm (192 mg m3) and kidney damage at 270 ppm (1731 mg m3) and above.In mice haematological effects were only seen at the top dose.

A 2-year inhalation study with rats exposed to approximately 0, 5, 25 or 125 ppm for 6 hours

per day, 5 days per week reported decreases in body weight, changes in haematology andblood biochemistry including markers of hepatotoxicity and nephrotoxicity at 25 ppm and a

significant decrease in survival at 125 ppm, predominantly due to liver tumours and/or

chronic nephropathy. In a parallel study, mice exposed to the same concentrations had asignificantly decreased survival rate, mainly due to liver tumours, when exposed to 25 or 125

ppm. A decrease in body weight gain, changes in haematology and blood biochemistry were

also observed at 25 ppm.

Ingestion

The liver and kidneys are also target organs for chronic oral exposure to carbon tetrachloride

in animal studies .Rats given carbon tetrachloride via gavage 5 days per week for 12 weeks had an increase inserum liver enzymes and mild liver damage (vacuolation) at 10 mg kg-1 bw day-1 and

cirrhosis was observed at 33 mg kg-1 bw day-1. No effects were seen at 1 mg kg-1 bw day-1.

In mice given carbon tetrachloride in corn oil 5 days a week for 90 days, changes in serumlevels of liver enzymes were seen at 12 mg kg-1 bw day-1 and above, together with

histopathological evidence of liver damage (fatty infiltration and necrosis). Similar to the

previous study, no effects were seen at 1.2 mg kg-1 bw day-1.

Oral exposure has also been associated with suppression of the immune system . One

study with mice given 50 mg kg-1 bw day-1 of carbon tetrachloride for 14 days (sufficient forliver toxicity) showed a reduced T-cell response to sheep red blood cells, and at 500 mg kg-1

bw day-1 a reduction in the absolute numbers of CD4+ and CD8- T-cells per spleen wasreported.

Genotoxicity

Carbon tetrachloride was not mutagenic in,but did induce DNA damage and mutations in

Escherichia coli . The results of in-vivo genotoxicity tests suggest that genotoxic effectsonly occur at doses that produce cytotoxicity . Although carbon tetrachloride has produced

CARBON TETRACHLORIDETOXICOLOGICAL OVERVIEW


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some effects on genetic material, such as in mammalian cells, the effects are considered to

be secondary to carbon tetrachloride toxicity and not from direct interaction with DNA [2, 5].

Thus, overall carbon tetrachloride is not regarded as genotoxic .Carbon tetrachloride has been extensively investigated in the Salmonella typhimurium invitroassay for gene mutation and negative results have been consistently obtained.

Negative results have also been obtained in assays for chromosome damage in hamsterovary cells, rat liver cells and human lymphocytes. DNA damage has been reported inE.Coli and it has been shown to induce intrachromosomal and mitotic recombination in yeast.

When investigated in-vivo, carbon tetrachloride did not induce chromosome aberrations in

bone marrow of mice or liver of rats. Nor did it induce micronuclei or unscheduled DNAsynthesis in the liver of rats and mice. DNA binding has been reported in the liver of rats,

mice and hamsters.

It has been suggested that the positive effects on carbon tetrachloride in a few assays are

explicable in terms of damage to nuclear protein or to DNA damage induced as a secondaryeffect to general toxicity .The weight of evidence indicates that carbon tetrachloride does not

have any significant genotoxic potential.

Carcinogenicity

Liver tumours have been produced in rats, mice and hamsters following carbon tetrachloride

exposure via oral, inhalation and subcutaneous administration . The doses inducing

liver tumours were higher than those causing liver cell toxicity, and therefore are consideredto arise secondary to toxic effects on the liver. IARC also noted one inhalation study in

mice that reported an increased incidence of a rare tumour in the adrenal glands. Overall,

IARC concluded that there is sufficient evidence for the carcinogenicity of carbon

tetrachloride in experimental animals.

Reproductive and developmental toxicity

There are a number of studies that have investigated the developmental toxicity of carbon

tetrachloride in pregnant rats, using the oral or inhalation route. There is also one study inmice using the oral route. In all cases adverse effects (limited to fetotoxicity rather than gross

malformations) were seen only at dose levels associated with maternal toxicity. It was

concluded that the available data suggest that the fetus is not preferentially sensitive to

carbon tetrachloride and the effects on fetal development and post-natal survival are likely to

be secondary to maternal toxicity .

Rats and mice were given injections of carbon tetrachloride into the portal vein or

into the spleen, and the localization of the resulting hepatocellular lesions was

studied. In contrast to some statements in the literature, and confirming some older

reports, damage was found to be limited to the periportal areas. A range of changes

leading to cell necrosis was found, which was frequently associated with pronouncedvasodilatation. These features were considered to be evidence in favour of a direct

hepatotoxic effect of carbon tetrachloride, and are difficult to reconcile with theview, formulated again recently by some workers, that carbon tetrachloride acts

indirectly on the liver cell by producing vasoconstriction.


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TOXIC EFFECT OF CARBON TETRACHLORIDE

It is injected directly into the portal vein. Since a similar blanching effect was seen following the

injection of hypertonic saline or glucose-an observation which I can confirm he attributed thischange to a " non-specific response to a strong irritant." It is

of interest, for the purpose of this paper, to draw attention to the discrepancy

inherent in the fact that on the one occasion when the contraction of vessels, whichis postulated to cause the centrilobular changes, is known to occur, the pathologicalchanges, as described here, take place in the periportal and not in the central region

of the liver lobule. Since the nature and the localization of these changes are highly

suggestive of a direct hepatotoxic effect of carbon tetrachloride, at least if given

intraportally, it is questionable whether an indirect effect of such an agent can be

invoked.

OBJECTIVEIt is well established that carbon tetrachloride (CCl4) is a typical hepatotoxin causing centrizonal

necrosis . As a result of extensive studies, the initial event in the rat given CCl4has been believed

to be lipid peroxidation of the endoplasmic reticulum of the liver cell initiated by trichloromethyl

radical generated by the reaction between CCl4and cytochrome P450. Based on these results,

CCl4has been assumed to be a typical poison causing severe oxidative stress and we will study

the oxidative stress produced by carbon tetrachloride by performing various enzyme and other

tests.

Oxidative stress induced by carbon tetrachloride in mice substantially decreases the increase inbody weight and relative testis weight. It also markedly increases the level of TBARS and

nitrites along with corresponding decrease in reduced glutathione and various antioxidant

enzymes in testis, i.e., catalase, peroxidase, superoxide dismutase and glutathione peroxidase.

Serum level of testosterone, luteinizing hormone and follicle stimulating hormone was

significantly decreased while estradiol and prolactin were increased with carbon tetrachloride

treatment.

CCl4-induced oxidative stress significantly cause reduced levels of glutathione (GSH), its

metabolizing enzymes and simultaneously cause the production of free radicals. This leads to

indirect genotoxicity and contribute to carcinogenicity.

the following tests were performed:-

1) Protein estimation by lowrys method

2) Lipid per oxidation

3) Catalase

4) Glutathione S transferase test


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5) ELISA

It is reported that the level of specifically determined lipid hydroperoxides , which was shown

to be a good indicator of oxidative stress in typical pathologic conditions such as vitamin E

deficiency , iron overload , diabetes , and thioacetamide intoxication, was increased by CCl4only

in mitochondria. Observations suggest that CCl4does not cause extensive lipid peroxidation aswidely accepted. To evaluate the oxidative stress caused by CCl4, changes in the concentration of

antioxidative vitamins C and E in the liver and plasma were measured in this study. carbon

tetrachloride (1 and 4 mM) produces a small elevation in M1dG adducts and DNA strand breaks

and increases in 8-oxodG were observed at the threshold of, and concomitant with, cytotoxicity.

Also the administration of CCl4increased lipid peroxidation and uric acid levels and inhibited

superoxide dismutase activity.

Protein concentrations were determined according to the method of Lowry et al. using bovine

serum albumin as the standard.

METHOD OF INJECTION

Carbon tetrachloride was tested for carcinogenicity in several experiments in mice

by oral and intrarectal administration and in rats by oral and subcutaneous

administration and by inhalation exposure; it was also tested in one experiment in

hamsters and one experiment in trout by oral administration. In various strains of

mice, it produced liver tumours, including hepatocellular carcinomas. In various

strains of rats, it produced benign and malignant liver tumours; and in oneexperiment with subcutaneous injection, an increased incidence of mammary

adenocarcinomas was observed. In hamsters and trout, increased incidences of

liver tumours were observed s2e9

For the estimation of effect of carbon tetrachloride on mice we took 6 mice out of

which four were taken as test and the rest two were taken as control.the effect of

carbon tetrachloride was determined by us in various estimation methods.

For carbon tetrachloride to be injected in the mice olive oil is used as a carrier.100

microlitre of olive oil and carbon tetrachloride in 1:3 is injected in intraperitonealcavity of four mice taken as test and 100 microlitre of olive oil is inject in two mice

taken as control.

The mices are now kept for 24 hours.


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Next day the mices are sacrificed. Mices are anesthetized with diethyl ether ans

then dissection is done and kidney, liver and testis are excised and the rest of the

part is disposed off.

The kidney, liver and testis need to be stored at low temperature so that their activity

is not lost. Now 200 mg of each sample is weighed. This sample is homogenizedusing a homoge nizer with tris HCl buffer prepared with ph 7.4.

200mg of of sample and tris HCl buffer is homogenized and the volume is made

upto 2 ml. Now take about 10% of homogenizer and centrifuge it at 10,000 rpm for

about 30 minutes. Now discard the pellet and store the supernatant in eppendoffs in

freezers.

PROTEIN ESTIM TION BY LOWRYS METHOD

AIM-estimation of protein in the 17 samples using standard plot

MATERIAL REQUIRED-

A) APPARATUS-

test tubes, burette, pipette, measuring cylinder, cuvette, spectrophotometer

B) REAGENTS

A. 2% Na2CO3 in 0.1 N NaOH

B. 2% NaK Tartrate in H2OC Copper sulphate solution-1%D. Lowrys reagent-98:1:1(A:B:C)

E. Phenol Reagent - 1 part Folin-Phenol [2 N] : 1 part water

BSA Standard - 1 mg/ mL(prepare fresh)

Bovine Serum Albumin: 5 mg in 5 mL of water


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PRINCIPLE-The Lowry protein assayis abiochemicalassayfor determining the total level ofproteinin a

solution. The total protein concentration is exhibited by a color change of the sample solution inproportion to protein concentration, which can then be measured usingcolorimetrictechniques.

It is named for the biochemistOliver H. Lowrywho developed the reagent in the 1940s. His

1951 paper describing the technique is the most-highly cited paper ever in the scientificliterature, cited over 200,000 times.

The principle behind the Lowry method of determining protein concentrations lies in thereactivity of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the

subsequent reduction of the Folin- Ciocalteay phosphomolybdic phosphotungstic acid to

heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic acid. The

concentration of the reduced Folin reagent is measured byabsorbanceat 750 nm.[

The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be

maintained at 10 - 10.5. The Lowry method is sensitive to low concentrations of protein. The

major disadvantage of the Lowry method is the narrow pH range within which it is accurate.However, we will be using very small volumes of sample, which will have little or no effect on

pH of the reaction mixture. A variety of compounds will interfere with the Lowry procedure.

These include some amino acid derivatives, certain buffers, drugs, lipids, sugars,salts, nucleic acids and sulphydryl reagents . It is noted that ammonium ions, zwitter ionic

buffers, nonionic buffers and thiol compounds may also interfere with the Lowry reaction. These

substances should be removed or diluted before running Lowry assays.

PROCEDURE-1) Prepare the chemicals as required.

2) Take 24 test tubes and label one as blank,6 as standard and 17 as test.3) In test tubes labeled as standard add 10,2,30,40,50,60 microlitre of standard

prepared and make up the volume to about 1ml with distilled water4) In blank take 1ml of distilled water

5) In test tubes labeled as test take add 10 microlitre of sample and make up the

volume to 1 ml with distillled water6) Now add 3ml of lowrys reagent in each test tube and incubate it for 30 minutes at

37 degree Celsius.

7) Add 300 microlitre of follins reagent to each test tube and again incunbate it for

30 minutes at 37 degree Celsius.8) Take reading at 620nm.

9) Plot the standard curve and estimate the amount of protein of tests from thestandard curve.
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OBSERVATIONS-

S.NO.

Std&test(ul)

Distilledwater(ul)

Lowrysreagent(ml)

Follinsreagent(ml)

O.D.

1 Blank - 1000 3.0 IncubateFor30Minutes

At 37degreecelsius

0.3 IncubateFor30Minutes

At 37

degreecelsius

0.0

2 S1 10 990 3.0 0.3 0.120

3 S2 20 980 3.0 0.3 0.162

4 S3 30 970 3.0 0.3 0.450

5 S4 40 960 3.0 0.3 0.192

6 S5 50 950 3.0 0.3 0.226

7 S6 60 950 3.0 0.3 0.270

8 T1 K 10 940 3.0 0.3 0.411

9 T1 L 10 990 3.0 0.3 0.339

10 T1 T 10 990 3.0 0.3 0.335

11 T2 K 10 990 3.0 0.3 0.542

12 T2 L 10 990 3.0 0.3 0.275

13 T2 T 10 990 3.0 0.3 0.197

14 T3 K 10 990 3.0 0.3 0.254

15 T3 L 10 990 3.0 0.3 0.332

16 T3 T 10 990 3.0 0.3 0.189

17 T4 K 10 990 3.0 0.3 0.42818 T4 L 10 990 3.0 0.3 0.273

19 T4 T 10 990 3.0 0.3 0.193

20 C1 K 10 990 3.0 0.3 0.422

21 C1 L 10 990 3.0 0.3 0.678

22 C2 K 10 990 3.0 0.3 0.093

23 C2 L 10 990 3.0 0.3 0.336

24 C2 T 10 990 3.0 0.3 0.201


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From graph-

Amount of protein in each test sample is determined from graph

S.NO. SAMPLE PROTEIN(ug/ul) For 10ugprotein(ul)

1 T1 K 9.1 1.098

2 T1 L 7.55 1.324

3 T1 T 7.5 1.33

4 T2 K 12 .833

5 T2 L 6.1 1.639

6 T2 T 4.3 2.325

7 T3 K 5.5 1.818

8 T3 L 7.3 1.369

9 T3 T 4.2 2.38010 T4 K 9.5 1.052

11 T4 L 6.0 1.66

12 T4 T 4.2 2.380

13 C1 K 9.3 1.075

14 C1 L 12.85 0.778

15 C2 K 2.0 5.00

16 C2 L 7.3 1.369

17 C2 T 4.4 2.272

PECAUTIONS-

1) Warming up time of 15 minutes must be givenbefore taking the readings.

2) Samples should be kept in crushed ice while taking the readings.

3) New tips must be taken for every sample.

C T L SE TEST

AIM-To calculate the enzyme activity by catalase method

MATERIAL REQUIRED-


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A) APPARATUS-


B) REAGENTS

1)Phosphate Buffer, 50mM(pH 7)

KH2PO4 (50mM) + NA2HPO4(50mM)

(40 ml) (60 ml)

Set pH with KOH.

KH2PO4 (50mM) 50mM= .680gm/100ml OR 1.02gm/150ml

Na2HPO4(50mM) 50mM= .709/100ml OR 1.063gm/150ml

2)0.75M Hydrogen Peroxide

THEORY-

Catalaseis a commonenzymefound in nearly all living organisms exposed to oxygen.

Itcatalyzesthe decomposition ofhydrogen peroxidetowaterandoxygen.[1]

It is a very important

enzyme in protecting the cell fromoxidative damagebyreactive oxygen species(ROS).

Likewise, catalase has one of the highestturnover numbersof all enzymes; one catalasemolecule can convert millions of molecules of hydrogen peroxide to water and oxygen each

second.[2]

The catalase enzyme serves to neutralize the bactericidal effects of hydrogen peroxide (13).

Catalase expedites the breakdown of hydrogen peroxide (H2O2) into water and oxygen (2H2O2 +

Catalase 2H2O + O2). This reaction is evident by the rapid formation of bubbles (2, 7).

PROCEDURE-

1) Prepare solution of .16 ml of H2O2in 100 ml of phosphate buffer

2) Cover the bottle with foil.

3) Check the O.D. of H2O2at 240 nm by taking 3ml in a cuvette. It should be around

0.5 and if it is less than that then add more of H2O2.

4) Take other test tubes and add 3ml of H2O2in each test tube.
http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Catalysthttp://en.wikipedia.org/wiki/Catalysthttp://en.wikipedia.org/wiki/Catalysthttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Waterhttp://en.wikipedia.org/wiki/Waterhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Catalase#cite_note-pmid14745498-1http://en.wikipedia.org/wiki/Catalase#cite_note-pmid14745498-1http://en.wikipedia.org/wiki/Catalase#cite_note-pmid14745498-1http://en.wikipedia.org/wiki/Oxidative_stresshttp://en.wikipedia.org/wiki/Oxidative_stresshttp://en.wikipedia.org/wiki/Oxidative_stresshttp://en.wikipedia.org/wiki/Reactive_oxygen_specieshttp://en.wikipedia.org/wiki/Reactive_oxygen_specieshttp://en.wikipedia.org/wiki/Reactive_oxygen_specieshttp://en.wikipedia.org/wiki/Turnover_numberhttp://en.wikipedia.org/wiki/Turnover_numberhttp://en.wikipedia.org/wiki/Turnover_numberhttp://en.wikipedia.org/wiki/Catalase#cite_note-2http://en.wikipedia.org/wiki/Catalase#cite_note-2http://en.wikipedia.org/wiki/Catalase#cite_note-2http://en.wikipedia.org/wiki/Catalase#cite_note-2http://en.wikipedia.org/wiki/Turnover_numberhttp://en.wikipedia.org/wiki/Reactive_oxygen_specieshttp://en.wikipedia.org/wiki/Oxidative_stresshttp://en.wikipedia.org/wiki/Catalase#cite_note-pmid14745498-1http://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Waterhttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Catalysthttp://en.wikipedia.org/wiki/Enzyme


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5) Now add 10 microlitre of sample in each test tube .

6) For each cuvette monitor the A240untill constant and add 10 microliter of sample.

7) Take 3 readings after the interval of 1 minutes.

OBSERVATIONS-

Reading of blank i.e. 3ml H2O2observed at 0.730

S. No. Samples(10

microliter)

H2O2(ml) O.D.

(Reading after 1

min)

O.D.

(Reading

after 2 min)

O.D.

(Reading after 3 min)

1. T1 K 3.0 0.771 0.600 0.518

2. T1 L 3.0 0.528 0.349 0.302

3. T1 Te 3.0 0.723 0.721 0.720

4. T2 K 3.0 0.680 0.576 0.493

5. T2 L 3.0 0.674 0.516 0.438

6. T2 Te 3.0 0.825 0.783 0.744

7. T3 K 3.0 0.749 0.642 0.604

8. T3 L 3.0 0.655 0.519 0.450

9. T3 Te 3.0 0.818 0.776 0.753

10. T4 K 3.0 0.772 0.766 0.763

11. T4 L 3.0 0.743 0.739 0.735

12. T4 Te 3.0 0.799 0.765 0.753

13. C1 K 3.0 0.854 0.679 0.578

14. C1 L 3.0 0.654 0.495 0.578

15. C1 Te 3.0 -- -- --

16. C2 K 3.0 0.724 0.717 0.717

17. C2 L 3.0 0.667 0.478 0.408


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18. C2 Te 3.0 0.829 0.811 0.795

RESULTS-

Extension coefficient=43.6mol-1cm-1

Enzyme activity=change in OD/min/ext coeff*mg of protein

Change in OD /min is calculated from the graph which is plotted between optical density and

time

S.NO. SAMPLE CHANGE INOD/MIN

PROTEIN INMICROLITRE

ENZYME ACTIVITY(mmol cm min

-1mg

-1)

1 T1 K 0.085 91 21.4

2 T1 L 0.125 75.5 37

3 T1 Te 0.010 75 3.054 T2 K 0.060 120 11.4

5 T2 L 0.100 61 37

6 T2 Te 0.035 43 18

7 T3 K 0.060 55 25

8 T3 L 0.075 73 22

9 T3 Te 0.030 42 16

10 T4 K 0.020 95 4.8

11 T4 L 0.020 60 7.6

12 T4 Te 0.030 42 16.3

13 C1 K 0.130 32 0.3214 C1 L 0.115 128.5 20.5

15 C2 K 0.010 20 11.46

16 C2 L 0.135 73 42

17 C2 Te 0.010 44 520

PRECAUTIONS-

1) Warming up time of 15 minutes must be givenbefore taking the readings.

2) Cuvette should be properly washed after each reading of sample.



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LIPID PEROXID TION TEST

-

AIM-To calculate the enzyme activity by LPO method

MATERIAL REQUIRED-

A) APPARATUS-


B) REAGENTS

STANDARDTetra Ethoxy Propane (TEP)( Range 2-10 nM)

22ul TEP100ml (100nM) 1ml in 100 ml(10nM)

10% TCA 1.25g/12.5ml dW

TBA(Thio Barbituric Acid) 0.67% (670mg in 100ml dW)

THEORY- Lipid peroxidationrefers to theoxidativedegradation oflipids.It is the process in

whichfree radicals"steal" electrons from the lipids incell membranes,resulting in cell damage.

This process proceeds by a free radicalchain reactionmechanism. It most oftenaffectspolyunsaturatedfatty acids,because they contain multiple double bonds in between

which liemethylene bridges(-CH2-) that possess especially reactivehydrogens.As with any

radical reaction, the reaction consists of three major steps: initiation, propagation, and

termination.

Hepatic injury through carbon tetrachloride (CCl(4)) induced lipid peroxidation is well known

and has been extensively used in the experimental models to understand the cellular

mechanisms behind oxidative damage and further to evaluate the therapeutic potential of drugs

and dietary antioxidants

PROCEDURE-

1) Firstly prepare the solutions needed.

2) Then thaw the sample.
http://en.wikipedia.org/wiki/Redoxhttp://en.wikipedia.org/wiki/Redoxhttp://en.wikipedia.org/wiki/Redoxhttp://en.wikipedia.org/wiki/Lipidhttp://en.wikipedia.org/wiki/Lipidhttp://en.wikipedia.org/wiki/Lipidhttp://en.wikipedia.org/wiki/Radical_(chemistry)http://en.wikipedia.org/wiki/Radical_(chemistry)http://en.wikipedia.org/wiki/Radical_(chemistry)http://en.wikipedia.org/wiki/Cell_membraneshttp://en.wikipedia.org/wiki/Cell_membraneshttp://en.wikipedia.org/wiki/Cell_membraneshttp://en.wikipedia.org/wiki/Chemical_reactionhttp://en.wikipedia.org/wiki/Chemical_reactionhttp://en.wikipedia.org/wiki/Polyunsaturatedhttp://en.wikipedia.org/wiki/Polyunsaturatedhttp://en.wikipedia.org/wiki/Fatty_acidshttp://en.wikipedia.org/wiki/Fatty_acidshttp://en.wikipedia.org/wiki/Fatty_acidshttp://en.wikipedia.org/wiki/Methylene_bridgehttp://en.wikipedia.org/wiki/Methylene_bridgehttp://en.wikipedia.org/wiki/Methylene_bridgehttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Methylene_bridgehttp://en.wikipedia.org/wiki/Fatty_acidshttp://en.wikipedia.org/wiki/Polyunsaturatedhttp://en.wikipedia.org/wiki/Chemical_reactionhttp://en.wikipedia.org/wiki/Cell_membraneshttp://en.wikipedia.org/wiki/Radical_(chemistry)http://en.wikipedia.org/wiki/Lipidhttp://en.wikipedia.org/wiki/Redox


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3) Now add 0.5ml supernatant sample and 0.5ml of Tris Hcl buffer in the 17 respective

eppendorfs.

4) Put these eppendorfs for 2 hour incubation at 370C.

5) Now add 750ul of TCA solution and 750ul of the above samples in another eppendorfs.

6) Spin the above samples at 3000rpm for 10 minutes.

7) Add 1ml of TBA solution and 1ml of supernatant from the centrifuged samples in the test

tubes.

8) Then put the test tubes in the water bath(preset at 1000C) for 30-45 minutes.

9) Now cool down the samples.

10) Observe the samples at 532nm under spectrophotometer and note O.D. of the samples.

OBSERVATIONSAND RESULTS-

Extension coefficient=1.56*105mmol

-1cm

-1

Enzyme activity= OD(3ml)/min/ext coeff*mg of protein

S.No. Samples O.D.

(2ml Sample)

O.D.

(3ml Sample)

ENZYME ACTIVITY

(mmol cm mg-1)

1. T1 K 0.620 0.401 0.56

2. T1 L 0.554 0.391 0.66

3. T1 Te 0.705 0.410 0.70

4 T2 K 0.481 0.300 0.32

5. T2 L 0.468 0.295 0.62

6. T2 Te 1.030 0.597 1.76

7. T3 K 0.532 0.310 0.72

8. T3 L 0.384 0.167 0.28

9. T3 Te 0.495 0.245 0.74

10. T4 K 0.615 0.340 0.4411. T4 L 1.03 0.51 1.08

12. T4 Te 0.223 0.140 0.426

13. C1 K 0.368 0.176 0.242

14. C1 L 0.495 0.315 0.30

15. C2 K 0.689 0.356 2.28

16. C2 L 0.155 0.091 0.44

17. C2 Te 0.470 0.256 0.26


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PRECAUTIONS-

1) Warming up time of 15 minutes must be given before taking the readings.

2) Cuvette should be properly washed after each reading of sample.3) New tips must be taken for every sample.

GLUT THIONE S TR NSFER SE

AIM- Measurement of glutathione s transferase activity by using CDNB as the substrate.

MATERIAL REQUIRED-

A) APPARATUS-


B) REAGENTS

1) CDNB(1 chloro 2,4-dinitrobenzene)=2mg/100ml of abs. alcohol

2)GSH(mM)= 3mg/10ml phosphate buffer {(1) & (2) to be prepared fresh.}

3)Phosphate Buffer[0.1M, pH6.5]

a)NaH2PO4.2H2O(0.1M)

b)Na2HPO4(0.1M) [128 ml of (a) + 72 ml of (b) & set pH 6.5]

THEORY-

Glutathione S-transferases in the small intestine function in detoxification of electrophilic

compounds ingested in foods, dietary supplements, and orally administered drug preparations.

Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes,

the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake

of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal

concentrations in the rat are about 250 M. The 250 M of extracellular GSH stimulated

conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte

preparations. However, an unexpected finding was that most of this stimulated activity did not

depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase

associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small

intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this


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detoxication enzyme in the extracellular mucus layer provides a novel mechanism for

preventing direct contact of potentially toxic dietary electrophiles with the intestinal

enterocytes.

Glutathione (GSH) is supplied to the lumen of the small intestine from bile transported from the

liver . The concentration of GSH in bile is in the millimolar range and supply of GSH to bileoccurs via a specific transport system, which has been extensively characterized in bile

canalicular membranes. GSH can also be provided from the diet, with fresh fruits and

vegetables providing on average the highest concentrations . Direct measurements of luminal

(jejeunal) GSH in rat show a concentration range of 60200 M in fasted animals that increases

to 120300 M in fed animals .

Intestinal epithelial cells contain members of the GST family that catalyze the reaction of GSH

with electrophilic compounds

Chemical carcinogens are generally classified as genotoxic or non-genotoxic. However, weakgenotoxicity at high concentrations is sometimes observed and interpretation is often

problematic. In addition, certain rodent carcinogens exert their effects at doses associated with

cytotoxicity and compensatory hyperplasia may be a contributing factor to tumourogenesis. We

hypothesise that certain substances, at high concentrations, can induce an oxidative stress via

the depletion of glutathione (GSH) and other antioxidant defences and that this may lead to

indirect genotoxicity, that could contribute to carcinogenicity. In support of this, human HepG2

cells treated with buthionine sulphoximine (BSO) to deplete GSH, exhibited DNA strand

breaks alongside elevated 8-oxodeoxyguanosine (8-oxodG) and malondialdehyde

deoxyguanosine (M1dG) adducts under conditions associated with lipid peroxidation. In female

rat hepatocytes, chloroform treatment resulted in a small dose-dependent increase in M1dGadducts (4 mM and above), DNA strand breakage (8 mM and above) and lipid peroxidation, in

the absence of any associated increase in DNA oxidation. GSH depletion only occurred in

association with cytotoxicity (20 mM; lactate dehydrogenase release). Alongside lipid

peroxidation, carbon tetrachloride (1 and 4 mM) produced a small elevation in M1dG adducts

and DNA strand breaks and increases in 8-oxodG were observed at the threshold of, and

concomitant with, cytotoxicity (4 mM). These effects may contribute to high dose genotoxicity

and carcinogenicity. Non-linearity in the dose response is expected on the basis of depletion of

antioxidants, and therefore, a pragmatic threshold for biologically relevant responses should

exist.

PROCEDURE-

1) Prepare the solutions as given in the requirements.

2) Take 17 test tubes and add 2.7 ml of phosphate buffer in each test tube.

3) Now add 0.1ml of CDNB

4) After this add about 0.1 ml of GSH in each test tube.


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5) Now add this solution in the cuvette in spectrophotometer and add 10 microlitre of sample

6) Now take the reading at 340 nm.

OBSERVATIONS-

S. No. Samples O.D.

(Reading after 1

min)

O.D.

(Reading after

2 min)

O.D.

(Reading after 3

min)

1. T1 K 0.269 0.254 0.259

2. T1 L 0.003 0.008 0.009

3. T1 Te 0.092 0.106 0.119

4. T2 K 0.011 0.010 0.012

5. T2 L 0.026 0.032 0.033

6. T2 Te 0.027 0.029 0.039

7. T3 K 0.108 0.112 0.124

8. T3 L 0.110 0.110 0.120

9. T3 Te 0.007 0.009 0.011

10. T4 K 0.094 0.106 0.130

11. T4 L 0.140 0.147 0.150

12. T4 Te 0.065 0.064 0.065

13. C1 K 0.122 0.140 0.137

14. C1 L 0.045 0.058 0.065

15. C2 K 0.021 0.023 0.027

16. C2 L 0.072 0.089 0.099

17. C2 Te 0.060 0.066 0.076

RESULT-

Extension coefficient=9.6 mmol-1

cm-1

Enzyme activity=change in OD/min/ext coeff*mg of protein

Change in OD /min is calculated from the graph which is plotted between optical density and

time

S.NO. SAMPLE CHANGE IN

OD/MIN

PROTEIN IN

MICROLITRE

ENZYME ACTIVITY

(mmol cm min-1

mg-1)

1 T1 K 0.020 91 22

2 T1 L 0.002 75.5 2.7

3 T1 Te 0.010 75 13

4 T2 K 0.001 120 0.86

5 T2 L 0.020 61 34


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6 T2 Te 0.0252 43 60.5

7 T3 K 0.010 55 17

8 T3 L 0.005 73 7.13

9 T3 Te 0.025 42 62

10 T4 K 0.035 95 38

11 T4 L 0.010 60 1712 T4 Te 0.001 42 2.4

13 C1 K 0.030 93 33

14 C1 L 0.010 128.5 8.1

15 C2 K 0.005 20 26

16 C2 L 0.015 73 21

17 C2 Te 0.015 44 35

PRECAUTIONS-

1) Warming up time of 15 minutes must be given before taking the readings.

4) Cuvette should be properly washed after each reading of sample.


ELIS

AIM-To perform ELISA

MATERIAL REQUIRED-

A) APPARATUS-


B) REAGENTS

1) Phosphate Buffered Saline

PBS(pH 7.2 , 0.1 M)

a)NaH2PO4 (M.Wt- 138) 0.1M=1.384 g/100ml

b)Na2HPO4(M.Wt-178gm) 0.1M=1.78g/100ml


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Nacl=0.9g/100ml. Add (a) to (b) to set pH

2) 0.06M Carbonate Buffer

a)NaHCO3= (8.4%) 0.1M b)Na2CO3=(10.6%) 0.1M

a(45.3) + b(18.0) Make upto 1 litre

For 100 ml-

a)NaHCO3= 2.1 g/25ml 4.53 ml + b)Na2CO3= 2.65 g/25ml 1.82 ml

3) Citrate Buffer (pH 5, 0.1M)

a)Citric Acid C6H8O7.H2O (525mg/25ml)

b)Na2HPO4.2H2O ( 445 mg/25ml)

Add a & b in equal volume for pH 5

4) Substrate

ABTS= 0.5mg/ml of Citrate Buffer

H2O2= 5 ul/10ml

[Prepare this just before adding to the plates]

5)tween 80

6)Carbonate buffer

THEORY-

Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with

the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-

assayed enzyme. ELISAs can provide a useful measurement of antigen or antibodyconcentration. There are two main variations on this method: The ELISA can be used to detect

the presence of antigens that are recognized by an antibody or it can be used to test for

antibodies that recognize an antigen.

ELISAs are performed in 96-well plates which permits high throughput results. The bottom of

each well is coated with a protein to which will bind the antibody you want to measure. Whole

blood is allowed to clot and the cells are centrifuged out to obtain the clear serum with


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antibodies (called primary antibodies). The serum is incubated in a well, and each well contains

a different serum (see figure below). A positive control serum and a negative control serum

would be included among the 96 samples being tested.

After some time, the serum is removed and weakly adherent antibodies are washed off with a

series of buffer rinses. To detect the bound antibodies, a secondary antibody is added to eachwell. The secondary antibody would bind to all human antibodies and is typically produced in a

rodent. Attached to the secondary antibody is an enzyme such as peroxidase or alkaline

phosphatase. These enzymes can metabolize colorless substrates (sometimes called chromagens)

into colored products. After an incubation period, the secondary antibody solution is removedand loosely adherent ones are washed off as before. The final step is the addition the enzyme

substrate and the production of colored product in wells with secondary antibodies bound.

When the enzyme reaction is complete, the entire plate is placed into a plate reader and the

optical density (i.e. the amount of colored product) is determined for each well. The amount of

color produced is proportional to the amount of primary antibody bound to the proteins on the

bottom of the wells.

TYPES OF ELISA

Direct ELISA

(1) Direct ELISAs involve attachment of the antigen to the solid phase, followed by an enzyme-

labeled antibody. This type of assay generally makes measurement of crude samples difficult,

since contaminating proteins compete for plastic binding sites.

Indirect ELISA

(2) Indirect ELISAs also involve attachment of the antigen to a solid phase, but in this case, theprimary antibody is not labeled. An enzyme-conjugated secondary antibody, directed at the first

antibody, is then added. This format is used most often to detect specific antibodies in sera.

Competitive ELISA

(3) The third type of ELISA is the Competition Assay, which involves the simultaneous addition

of 'competing' antibodies or proteins. The decrease in signal of samples where the second

antibody or protein is added gives a highly specific result.

Sandwich ELISA

(4) The last type of assay is the sandwich ELISA. Sandwich ELISAs involve attachment of a

capture antibody to a solid phase support. Samples containing known or unknown antigen are

then added in a matrix or buffer that will minimize attachment to the solid phase. An enzyme-

labeled antibody is then added for detection.

HMOX1(heme oxygenase (decycling) 1) is ahumangenethat encodes for the enzymeheme

oxygenase 1. Heme oxygenase is an essential enzyme inheme catabolism,it cleaveshemetoformbiliverdin.
http://en.wikipedia.org/wiki/Humanhttp://en.wikipedia.org/wiki/Humanhttp://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Heme_oxygenasehttp://en.wikipedia.org/wiki/Heme_oxygenasehttp://en.wikipedia.org/wiki/Heme_oxygenasehttp://en.wikipedia.org/wiki/Heme_oxygenasehttp://en.wikipedia.org/w/index.php?title=Heme_catabolism&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Heme_catabolism&action=edit&redlink=1http://en.wikipedia.org/w/index.php?title=Heme_catabolism&action=edit&redlink=1http://en.wikipedia.org/wiki/Hemehttp://en.wikipedia.org/wiki/Hemehttp://en.wikipedia.org/wiki/Hemehttp://en.wikipedia.org/wiki/Biliverdinhttp://en.wikipedia.org/wiki/Biliverdinhttp://en.wikipedia.org/wiki/Biliverdinhttp://en.wikipedia.org/wiki/Biliverdinhttp://en.wikipedia.org/wiki/Hemehttp://en.wikipedia.org/w/index.php?title=Heme_catabolism&action=edit&redlink=1http://en.wikipedia.org/wiki/Heme_oxygenasehttp://en.wikipedia.org/wiki/Heme_oxygenasehttp://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Human


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Heme oxygenase, an essential enzyme in heme catabolism, cleaves heme to form biliverdin,

which is subsequently converted to bilirubin by biliverdin reductase, and carbon monoxide, a

putative neurotransmitter. Heme oxygenase activity is induced by its substrate heme and byvarious nonheme substances. Heme oxygenase occurs as 2 isozymes, an inducible heme

oxygenase-1 and a constitutive heme oxygenase-2. HMOX1 and HMOX2 belong to the heme

oxygenase family

The 70 kilodalton heat shock proteins(Hsp70s) are a family of conserved ubiquitously

expressedheat shock proteins.Proteins with similar structure exist in virtually all livingorganisms. The Hsp70s are an important part of the cell's machinery for protein folding, and help

to protect cells from stress.In cooperation with other chaperones, Hsp70s stabilize preexistentproteins against aggregation and mediate the folding of newly translated polypeptides in the

cytosol as well as within organelles. These chaperones participate in all these processes throughtheir ability to recognize nonnative conformations of other proteins. They bind extended peptide

segments with a net hydrophobic character exposed by polypeptides during translation and

membrane translocation, or following stress-induced damage. In case of rotavirus A infection,

serves as a post-attachment receptor for the virus to facilitate entry into the cell.

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)or ABTSis chemical compound usedto observe thereaction kineticsof specificenzymes.A common use for it is in theenzyme-

linked immunosorbent assay(ELISA)to detect for binding of molecules to each other.

It is commonly used as asubstratewithhydrogen peroxidefor aperoxidaseenzyme or alone

with bluemulticopper oxidaseenzymes such aslaccaseorbilirubin oxidase.Its use allows thereaction kinetics ofperoxidasesthemselves to be followed. In this way it also can be used to

indirectly follow the reaction kinetics of anyhydrogen peroxide-producing enzyme, or to simply

quantify the amount of hydrogen peroxide in a sample.

PROCEDURE-

1) Coat the well with Ag diluted in carbonated buffer (100 ul/well).Keep it overnight at 40C in

humid chamber.

2) Flick the plate to throw the Ag and blockthe unbound space with 1% BSA in 0.1M

PBS(100ul/well).

3) Incubate at 370C ,1 hour.

4) Wash the plate (after flicking) with PBS-Tween 20 (125 ul/250ml of PBS) by filling, and

then flicking.[Each washing should be for atleast 3 minutes and give atleast 3-5

washings]
http://en.wikipedia.org/wiki/Heat_shock_proteinhttp://en.wikipedia.org/wiki/Heat_shock_proteinhttp://en.wikipedia.org/wiki/Heat_shock_proteinhttp://en.wikipedia.org/wiki/Enzyme_kineticshttp://en.wikipedia.org/wiki/Enzyme_kineticshttp://en.wikipedia.org/wiki/Enzyme_kineticshttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzyme-linked_immunosorbent_assayhttp://en.wikipedia.org/wiki/Enzyme-linked_immunosorbent_assayhttp://en.wikipedia.org/wiki/Enzyme-linked_immunosorbent_assayhttp://en.wikipedia.org/wiki/Enzyme-linked_immunosorbent_assayhttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/Substrate_(biochemistry)http://en.wikipedia.org/wiki/Substrate_(biochemistry)http://en.wikipedia.org/wiki/Substrate_(biochemistry)http://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Peroxidasehttp://en.wikipedia.org/wiki/Peroxidasehttp://en.wikipedia.org/wiki/Peroxidasehttp://en.wikipedia.org/wiki/Multicopper_oxidasehttp://en.wikipedia.org/wiki/Multicopper_oxidasehttp://en.wikipedia.org/wiki/Multicopper_oxidasehttp://en.wikipedia.org/wiki/Laccasehttp://en.wikipedia.org/wiki/Laccasehttp://en.wikipedia.org/wiki/Laccasehttp://en.wikipedia.org/wiki/Bilirubin_oxidasehttp://en.wikipedia.org/wiki/Bilirubin_oxidasehttp://en.wikipedia.org/wiki/Bilirubin_oxidasehttp://en.wikipedia.org/wiki/Peroxidaseshttp://en.wikipedia.org/wiki/Peroxidaseshttp://en.wikipedia.org/wiki/Peroxidaseshttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Peroxidaseshttp://en.wikipedia.org/wiki/Bilirubin_oxidasehttp://en.wikipedia.org/wiki/Laccasehttp://en.wikipedia.org/wiki/Multicopper_oxidasehttp://en.wikipedia.org/wiki/Peroxidasehttp://en.wikipedia.org/wiki/Hydrogen_peroxidehttp://en.wikipedia.org/wiki/Substrate_(biochemistry)http://en.wikipedia.org/wiki/ELISAhttp://en.wikipedia.org/wiki/Enzyme-linked_immunosorbent_assayhttp://en.wikipedia.org/wiki/Enzyme-linked_immunosorbent_assayhttp://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzyme_kineticshttp://en.wikipedia.org/wiki/Heat_shock_protein


27/29

5) Dilute the primary Ab in PBS-Tween containing 1% BSA.

6) Add 100ul to each well, incubate for 2 hours at 370C .

7) Flick and wash with PBS_Tween.

8) Dilute the Secondary Ab in PBS-Tween containing 1% BSA & 100ul/well.

9) Keep at 370C for 2 hours and then flick & wash.

10) PBS-Tween act as inhibitor of ABTS ,therefore,give one last washing with ddH2O before

adding ABTS.

11) Prepare substrate, add100ul to each well.

12) Keep in dark for 30 minutes.

13) Quantitative colour reaction at 405 nm.

OBSERVATION-


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S.No. NAME SAMPLE BUFFER(microliter)

1 C1 K 12.9 1200

2 C2 L 16.428 1200

3 C2 T 27.264 1200

4 T4 K 12.624 12005 T4 L 19.84 1200

6 T4 TE 28.56 1200

RESULTS-


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