Enzyme assays Salivary Amylase, SGOT, SGPT etc

Compiled by | Dr. S. K. HAYATH BASHA & Mr. V. MAGENDIRA MANI 1

ASSAY OF SALIVARY AMYLASE

AIM

To assay salivary amylase to determine its Optimum pH, optimum temperature, Specific activity

PRINCIPLE

α- amylase catalyses the hydrolysis of α-(1-4) glycosidic linkage and produces reducing sugar

like maltose, which is then coupled with 3, 5 – dinitro salicylic acid in alkali medium. It

produces an orange coloured complex. The intensity of the colour developed can be measured at

540 nm, which is directly proportional to the activity of the enzyme

REAGENTS REQUIRED

1. Substrate - 1% starch solution (1 gm of starch in 100 ml of water)

2. BIFFER - PHOSPHATE BUFFER

Solution A : 0.2m Disodium hydrogen phosphate

28.392 gm of disodium hydrogen phosphate is dissolved in 1 litre of distilled water.

Solution B : 0.2m sodium dihydrogen phosphate.

31.202 gm of sodium dihydrogen is dissolved in 1 litre of distilled water

BUFFER COMPOSITION

pH Solution A (ml) Solution B (ml) Dilution

5.8 8.0 92.0 200 ml of dis. water

6.4 26.5 73.5 200 ml of dis. Water

7.0 61.0 39.0 200 ml of dis. water

7.6 87.0 13.0 200 ml of dis. Water

8.0 94.7 5.3 200 ml of dis. water

3. Activator – 1% sodium chloride solution: 1 gm in 100 ml of dis water

4. 2N NaOH - 8 gm in 100 ml dis water

5. Salivary amylase - 1 in 20 dilution of salivary amylase in buffer


6. COLOURING AGENT : DNSA Dinitro salicylic acid

1 gm of 3, 5 - dinitro salicylic acid, 30 gm of sodium potassium tartarate, 1.6 gm of

sodium hydroxide is dissolved in dis water and finally made up to 100 ml using dis water.

7. Standard BSA - 100 mg of Bovine serum Albumin dissolved in 100 ml of

dis water

8. Lawry’s reagent - Alkaline copper tartarate reagent

Solution A - 12 gm of sodium carbonate and 2.4 gm of sodium hydroxide diluted to 600

ml of dis water.

Solution B - 100 mg of sodium potassium tartarate and 50 mg of copper sulphate

diluted in to 100 ml.

9. Folin’s phenol reagent

10. Maltose solution - 200 mg of maltose dissolved in 100 ml of water (2 mg/ml)


EFFECT OF pH ON SALIVARY AMYLASE

S.No

Contents in the test

tubes

Blank pH: 5.8 pH: 6.4 pH: 7.0 pH: 7.6 pH: 8.0

B B1 T1 B2 T2 B3 T3 B4 T4 B5 T5

1

Volume of the Buffer

(ml)

- 1.5 ml

2

Volume of substrate

(ml)

- 1.5 ml

3

Volume of activator

NaCl (ml)

1.0 ml

4 Volume of Dis water 4.0 1.0 ml

Pre inculpate all the test tubes at 37°C for 15 min

5

Volume of Enzyme

(ml)

- - 0.5 - 0.5 - 0.5 - 0.5 - 0.5

6 Volume of NaOH 0.5 ml

7

Volume of Enzyme

(ml)

- 0.5 - 0.5 - 0.5 - 0.5 - 0.5 -

8 Volume of DNSA(ml) 0.5 ml

Kept all the test tubes in boiling water bath for 10 min

9

OD measured at 540

nm

10

Difference in OD

Test - Blank


EFFECT OF pH ON SALIVARY AMYLASE

AIM:-

To determine the optimum PH of salivary amylase

PROCEDURE:-

Take five sets of clean test tubes marked as B1T1, B2T2, B3T3, B4T4, & B5T5. Add

1.5 ml of substrate to all the test tubes. Then add 1.5 ml of buffer prepared at various pH 6.0, 6.4,

6.8, 7.2 & 7.6 to the test tubes B1T1 to B5T5 respectively. Add 1 ml of activator (NaCl) to all

the test tubes. Make up the volume with 1ml of distilled water is added from B1T1 to B5T5.

Then all the test tubes are pre incubated at 37 °C for 5 min. Then add 0.5 ml of enzyme to test

tubes and marked as T1 & T5 and incubated at 37 °C for 10 min. Then add 1 ml of NaOH to all

test tubes including reagent blank. After that 0.5 ml of enzymes is added to blank marked as B1

to B5. Finally 0.5 ml of colouring reagent is added to all the test tubes including blank. The

reagent blank contains 4.5 ml of distilled water, 1 ml of NaCl, 0.5 ml of NaOH and 0.5 ml of

DNSA all the contents of the test tubes are mixed well.

Then the test tubes kept in boiling water bath for 10 min (or) until colour developed cooled. The

intensity of the colour developed is read at 540 nm green filter. The difference between test and

blank is noted and a graph is drawn pH against the optical density.

RESULT:-

The optimum PH of the enzyme salivary amylase is ___________.


EFFECT OF TEMPERATURE ON SALIVARY AMYLASE

S.No


tubes

Blank 0°C 37° C 60° C 90° C

B B1 T1 B2 T2 B3 T3 B4 T4

1


(ml)

- 1.5 ml

2

Volume of substrate

(ml)

- 1.5 ml

3

Volume of activator

NaCl (ml)

1.0 ml

4 Volume of Dis water 5.0 1.0 ml


5

Volume of Enzyme

(ml)

- - 0.5 - 0.5 - 0.5 - 0.5


7

Volume of Enzyme

(ml)

- 0.5 - 0.5 - 0.5 - 0.5 -



9

OD measured at 540

nm

10

Difference in OD

Test - Blank


EFFECT OF TEMPERATURE ON SALIVARY AMYLASE

AIM:-

To determine the optimum temperature of salivary amylase

PROCEDURE:-

Take four different test tubes sets marked as B1T1, B2T2, B3T3, and B4T4 respectively.

To all the test tubes add 1.5 ml of substrate and 1.5 ml of buffer (pH- 6.8). Then 1 ml of activator

NaOH and 1 ml of Dis water is added to all the test tubes. Pre incubated all the test tubes for 5

min. Then 0.5 ml of enzyme is added to test (T1-T4) only. The test tubes are than incubated at

following respectively temperature 0 °C, 37 °C, 60 °C and 90 °C for 10 min. After 15 min the

reaction is arrested by adding 0.5 ml NaOH to all the test tubes. Then 0.5 ml of enzyme is added

to the test tubes are marked as (B1-B4) blank. To all the test tubes are add 0.5 ml of dinitro

salicylic acid reagent and heat in boiling water bath for 10 min. Then the intensity of colour-

developed is marked at 540 nm.

A graph is drawn between the temperature plotted on x-axis and along y-axis. From the

graph the optimum temperature of enzyme is calculated.

RESULT:-

The optimum temperature of salivary amylase is at ________.


EFFECT OF SUBSTRATE CONCENTRATION ON SALIVARY AMYLASE

S.No


tubes

Blank SUBSTRATE CONCENTRATION

B B1 T1 B2 T2 B3 T3 B4 T4 B5 T5

1 Volume of substrate - 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.5

2 Concentration 1000 2000 3000 4000 5000

3


(ml)

- 1.5 ml

4

Volume of activator

NaCl (ml)

0.5 ml

4 Volume of Dis water 3.0 2.9 2.9 2.8 2.8 2.7 2.7 2.6 2.6 2.5 2.5


5

Volume of Enzyme

(ml)

- - 0.5 - 0.5 - 0.5 - 0.5 - 0.5


7

Volume of Enzyme

(ml)

- 0.5 - 0.5 - 0.5 - 0.5 - 0.5 -



9

OD measured at 540

nm

10

Difference in OD

Test - Blank


EFFECT OF SUBSTRATE CONCENTRATION ON SALIVARY AMYLASE

AIM:-

To determine the effect of substrate concentration on salivary amylase

PROCEDURE:-

Take five sets of clean test tubes marked as B1T1, B2T2, B3T3, B4T4, & B5T5.

0.1, 0.2, 0.3, 0.4, 0.5 ml of substrate solution is pipette out into all the test tubes. 1.5 ml of buffer

(pH=6.8) followed by 0.5ml of activator added to each tubes and volume of all tubes made upto

5 ml using distilled water. 0.5 ml if activator 5 ml of distilled water added to the tubes labelled as

Rb (reagent blank). All tubes are pre incubated for 5 min at 37 ° C add 0.5 ml of enzyme to the

tubes T1 to T5 only. Incubate all tubes at 37 ° C for 10 min. Now add 0.5 ml inhibitor NaOH to

each tube followed 0.5 ml of enzyme to test tubes B1 to B5 only. Now add 0.5 ml of DNSA to

all tubes and kept in boiling water bath for 1 min (until color developed). The intensity of colour

is read at 540 nm using green filter. A graph is drawn in the substrate concentration were as O.D

RESULT:-

Effect of substrate concentrated of salivary amylase (KM = ½ Vmax) =

__________________________________moles.


ASSAY OF SGOT (SERUM GLUTAMATE OXALO ACETATE TRANSAMINASE)

or AST (ASPARTATE AMINO TRANSFERASE)

S.NO Contents taken in the test

tubes (ml)

Blank

(B)

Working standard solution

S1 S2 S3 S4 S5

1 Working Standard solution

(ml)

-- 0.1 0.2 0.3 0.4 0.5

2 Concentration (µg) -- 0.2 0.4 0.6 0.8 1.0

4 Volume of substrate (ml) 0.2 ml

5 Distilled water (ml) 1.0 0.9 0.8 0.7 0.6 0.5

6 Volume of DNPH (ml) 1 ml

The contents of all the test tubes are mixed well and

allowed to stand for 15 minutes at room temperature

7 Volume of NaOH (ml) 10 ml



6 Optical Density at 540 nm

using green filter

--


ASSAY OF SGOT (SERUM GLUTAMATE OXALO ACETATE TRANSAMINASE)

or AST (ASPARTATE AMINO TRANSFERASE)

AIM:-

To assay the activity of serum glutamate oxalo acetate transaminase (SGOT) (or) Aspartate

amino transferase (AST)

PRINCIPLE:-

Amino transferase are groups of enzyme that catalysis the transfer of amino group of an amino

acid to keto acid they use pyriodoxal phosphate as cofactor. Two important clinical amino

transferases are

1) AST (or) SGOT

2) ALT (or) SGPT

The reaction of SGOT is


S. No Contents present in the test tube (ml) Blank (ml) Test (ml)

1 Volume of the substrate 1.0 1.0

Incubate at 37°C for 5 min

2 Volume of serum - 0.2


3 Volume of DNPH 1.0 1.0

4 Volume of serum 0.2 -

Mix it well and kept at room

temp for 10 min

5 Volume of 0.4 N NaOH 10 10

6


temp for 10 min

7 OD at 540 nm

8 Difference in OD


The activity of SGOT is determined by measuring the colour of intensity of 2,4-dinitro phenyl

hydrazine (DNPH) (Brown colour). This colour is found by the reaction between 2-4 dinitro

phenyl hydrazine and the keto acid in alkaline medium.

NOTE: DNPH gives more colour with oxalo acetate and pyruvate than with oxaloglutarate.

REAGENT REQUIRED:-

1) PHOSPHATE BUFFER( PH – 7.4 : 0.1M)

Solution A:

14.196 gm of disodium hydrogen phosphate (Na₂HPo₄) is weighed accurately and made upto 1

litre using distilled water.

Solution B:

15.601g of sodium dihydrogen phosphate is weighed and made upto 1 litre using distilled water.

Mix 840ml of Solution A with 160 ml of Solution B which gives a PH value of 7.4 check the PH

if needed by adjusting it with either one according (Na₂HPO₄) is NaHPo₄ for increasing the PH

and NaH₂PO₄ for decreasing the PH.

2) SUBSTRATE FOR SGOT (BUFFERED SUBSTRATE FOR SGOT)

2.66 gm of L- aspartic acid and 30 mg α- keto glutarate are weighed and dissolved in 2.5 ml of

1N NaOH and adjust the PH to 7.4 by adding NaOH solution drop by drop. The volume is made

upto 100 ml with phosphate buffer.

3) DINITROPHENYL HYDRAZINE:-

50 mg of DNPH is dissolved in 250 ml using 1N HCL and warmed is necessary.

4) 0.4 N NaOH:-

1.6 g of NaOH in 100 ml of distilled water.


Calculation

Difference in OD between test and Blank - Test – Blank

______________ OD

________OD of sample corresponds to __________________ µmoles of product

Enzyme activity

___________ µmoles of product and product liberated / min/ lit

Activity of SGOT

Concentration of Product X 1000

Volume of serum incubation time (min)

=__________________

Expression in Karman unit

1 karman unit - 0.483/lit

_ -____________/0.483

= _____________


5) STOCK STANDARD SOLUTION:-

220 mg of sodium pyruvate is dissolved in phosphate buffer and the volume is made

upto 100 ml (20 mm/lit or 20 mm/ml)

6) WORKING STANDARD SOLUTION:-

10 ml of stock standard solution is diluted to 100ml with phosphate buffer.

PROCEDURE:-

Construction of calibration curve

Working standard solution of sodium pyruvate is taken in the range of 0.1 ml - 0.5 ml into

5 different test tubes marked as S1- S5 respectively. Add 0.2 ml of distilled water to all the test

tubes including blank. Make up the volume in all test tubes in 1.2 ml of appropriate buffered

substrate solution. This is followed by addition of 1 ml of DNPH. Mix the content of all the tube

and keep it for 15 min at room temperature. Now 10 ml of 0.4N NaOH is added all the tubes

mixed well and allowed to stand for 5 min at room temperature.

ASSAY OF SGOT:-

1ml of the buffer substrate is taken in 2 different test tubes marked as B & T incubate

the tubes for 5min at 37 C. Now add 0.2 ml of serum of the tube T only. Mix well and incubate

at 37 C for 60 min and 1ml of DNPH reagent to both the test tubes. Now 0.2 ml of serum is

added to the tube mix the content and keep the room temperature for 15 min. Now add 0.4 ml

NaoH to both the tubes after 5 min measure the OD at 520nm.

NORMAL VALUES OF SGOT:-

SGOT - 7 - 21 IU (INTERNATIONAL UNIT)

1 IU - 1 µmoles of pyruvate equivalent formed /min / liter of serum of 37 C.


RESULT:-

The activity of SGOT =

___________ μ moles of oxaloacetate liberated/min /lit

(Or)

___________ IU /litre (or) ------------------ karmen unit / litre.


ASSAY OF SERUM GLUTAMATE PYRUVATE TRANSAMINASE (SGPT) or ALT

ALANINIE AMINO TRANSAMINASE


tubes (ml)

Blank

(B)


S1 S2 S3 S4 S5


(ml)

-- 0.1 0.2 0.3 0.4 0.5

2 Concentration (µg) -- 0.2 0.4 0.6 0.8 1.0

4 Volume of substrate (ml) 0.2 ml

5 Distilled water (ml) 1.0 0.9 0.8 0.7 0.6 0.5

6 Volume of DNPH (ml) 1 ml



7 Volume of NaOH (ml) 10 ml




using green filter

--


ASSAY OF SERUM GLUTAMATE PYRUVATE TRANSAMINASE (SGPT) or ALT

ALANINIE AMINO TRANSAMINASE

AIM:-

To assay the activity of SGPT (Serum Glutamate Pyruvate Transaminase) or ALT

(Alaninie Amino Transaminase).

PRINCIPLE:-

Amino transferase are groups of enzyme that catalysis the transfer of an amino group from α-

amino acid to keto acid. They use pyriodoxal phosphate as its cofactor. Two important clinical

amino transferases are

1) SGPT (or) ALT

2) SGOT (or) AST


The activity of SGPT is determined by measuring the colour of intensity of 2,4-dinitro phenyl

hydrazine (DNPH)(Brown). Which is formed by the reaction between DNPH gives more colour

to oxaloacetate then pyruvate.

REAGENT REQUIRED:-

1) PHOSPHATE BUFFER( PH – 7.4 : 0.1Mol)

Solution A:

14.196 gm of disodium hydrogen phosphate (Na₂HPo₄) is weighed accurately and made

upto 1 litre using distilled water.

Solution B:

15.601 gm of sodium dihydrogen phosphate is weighed and made upto 1 litre using

distilled water. Mix 840 ml of solution A with 160 ml of solution B which gives a p H value of

7.4 check the PH if needed by adjusting it with either one according (Na₂HPO₄) increase pH

and NaH₂PO₂ decrease pH.

2) SUBSTRATE FOR SGPT (BUFFERED SUBSTRATE FOR SGPT)

1.78gm of α- L- alanine and 30 mg α- keto glutarate is dissolved in 20 ml of phosphate

buffer containing 1.25 ml of 0.4 N sodium hydroxide. Adjust the pH to 7.4. Then the volume is

made upto 100 ml with phosphate buffer.

[Concentration = 200 millimole/litre]

3. DINITROPHENYL HYDRAZINE(DNPH):-

50 mg of DNPH is dissolved in 250ml using 1N HCL and if necessary warm.

4) 0.4 N NaOH:-

1.5 gm of NaOH in 100 ml of distilled water.


S.No Contents present in the test tube (ml) Blank (ml) Test (ml)





3 Volume of DNPH 1.0 1.0



temp for 10 min

5 Volume of 0.4 N NaOH 10 10

6


temp for 10 min

7 OD at 540 nm

8 Difference in OD



220 mg of sodium pyruvate is dissolved in phosphate buffer and made upto 100 ml

(Concentration: 20 millimole/lit or 20 micromole/ml)


10 ml of stock standard solution is diluted to 100 ml with phosphate buffer.

(Concentration: 2millimole/litre (or) 20 micro mole/ml)

PROCEDURE:-


Working standard solution of sodium pyruvate is taken in the range of 0.1 ml - 0.5 ml in

the test tubes marked as S1- S5 respectively. Add 0.2ml of distilled water in all the test tubes

including blank. Make up the volume in all test tubes to 1.2 ml with appropriate buffer substrate

solution. This is followed by of 1 ml of DNPH. The contents in all the test tube are mixed well

and kept it for 15min at room temperature. Now 10 ml of 0.4 N NaOH is added to all the test

tubes. Then mix well and allow standing for 5 min at room temperature. Measure the OD at 520

nm. Construct a calibration curve between the concentration and OD of the pyruvate.

ASSAY OF SGPT:-

1ml of buffer substrate (α-L-Alanine and α-ketoglutarate) is taken in two test tubes

mixed as B and T incubate the test for 5min at 37 °C. Now 0.2 ml of serum of the test tube

marked as Test (T) only and mixed well. Then it is incubated at 37°C for 60 min. Then add 1ml

of serum to the test tube marked as Blank (B) only. Mixed the content and keep it for 15min.

Then add 10 ml of 0.4N NaOH to both the test tube. Mixed well and keep it at room temperature

for 5min and measure the OD at 520nm.

NORMAL VALUES OF SGPT:-

6 -21 IU (INTERNATIONAL UNIT)

1 IU- 1Micromole of pyruvate (or) pyruvate equivalent formed /min 1litre of serum at 37 c.


Calculation


______________ OD


Enzyme activity


Activity of SGOT

Concentration of Product X 1000

Volume of serum incubation time (min)

=__________________


1 karman unit - 0.483/lit

_ -____________/0.483

= _____________


RESULT:-

Activity of SGPT 26.66 micromoles of pyruvate/min/hrs liberated.

(or)

=___________ IU /hr

(or)

=___________ karmen units.


ASSAY OF SERUM ALKALINE PHOSPHATASE BY MORY’S METHOD


tubes (ml)

Blank

(B)


S1 S2 S3 S4 S5


(ml)

-- 0.5 1.0 1.5 2.0 2.5

2 Concentration (µg) -- 5 10 15 20 25

4 Volume of buffered substrate

(ml) 1.0 ml

5 Distilled water (ml) 2.5 2.0 1.5 1.0 0.5 -

6 Volume of MgCl2 (ml) 0.1 ml

7 Volume of Folin’s Phenol

reagent 1.0 ml

7 Volume of 15 % Na2CO3 (ml) 1.0 ml




using red filter

--


ASSAY OF SERUM ALKALINE PHOSPHATASE BY MORY’S METHOD

AIM:-

To estimate the amount of alkaline phosphatase by Mory’s method modified by King

(1965) using Disodium phenyl phosphate as substrate.

PRINCIPLE:-

Alkaline phosphatase hydrolysis a variety of phosphate mono esters at alkaline pH to liberate

inorganic phosphate. The serum enzyme has its origin from liver, intestine, bone, spleen and

kidney. Alkaline phosphatase act on Disodium phenyl phosphate and liberate phenol which is

proportional to enzyme activity. Phenol reacts with Folin’s phenol reagent in the presence of

alkali to give blue colour. Which is measured at 640 nm using red filter.

REAGENT REQUIRED:-

1) SUBSTRATE FOR ALP (BUFFERED SUBSTRATE FOR ALP)

0.01 M Disodium phenyl phosphate

Exactly 0.54 mg of Disodium phenyl phosphate is dissolved in Bicarbonate buffer. (Bicarbonate

buffer: 0.1 M; 0.36 g of sodium carbonate and 3.3 g of sodium hydrogen carbonate are dissolved

in distilled water and made upto one liter which gives pH.10)


S.No Contents present in the test tube (ml) Blank (ml) Test (ml)



2 Activator MgCl2 ml 0.1 0.1



4 Volume of Folin’s phenol reagent 1.0 1.0


Mix it well and centrifuge it for

15 min

6 Volume of supernatant (ml) 4 4

7 Volume of 15 % Na2CO3 1 1

6 OD at 640 nm using red filter

8 Difference in OD


2) 0.1 M MgCl2

Activator 2.0331 g in 100 ml distilled water

3) Folin’s phenol reagent

1:2 dilution, 1 ml of Folin’s phenol reagent is added to 2 ml of dis. Water

4) 15 % Sodium carbonate

15 g Sodium carbonate in 100 ml dis water


100 mg phenol is weighed is dissolved in 100 ml dis water

(Concentration: 1 milli gram/1 ml)


1 ml of stock standard solution is diluted to 100 ml standard flask using dis water.

(Concentration: 10 micro gram/1 mll)

PROCEDURE:-


Working standard solution of phenol is taken in the range of 0.5 ml - 2.5 ml in the test

tubes marked as S1- S5 respectively. Now add 1.2 ml of appropriate buffer substrate solution to

all test tubes. The volume of all the test tubes are made upto 2.5 ml using dis. Water. Now add

0.1 ml of activator. This is followed by of 1 ml of Folin’s phenol reagent to all test tubes. The

contents in all the test tube are mixed well and 1 ml of 15 % Na2CO3 is added to all the test

tubes. Then mix well and allow standing for 10 min at room temperature. Measure the OD at 640

nm. Construct a calibration curve between the concentration and OD of the phenol.


Calculation


______________ OD


Enzyme activity


Activity of alkaline phosphatase

µmoles of phenol liberated x 1000 x 9 x 1

Molecular weight of phenol volume of serum volume of incubation time

Supernatant

= __________________ micro moles of phenol liberated / min/ liter


1 karman unit - /0.483

_ ____________


ASSAY OF ALKALINE PHOSPHATASE:-

6 ml of buffer substrate is taken in two test tubes mixed as B and T. All the test tubes are

incubated at 37 °C for 5 min. Then 0.1 ml of MgCl2 is added to both the tubes followed by

addition of 0.1 ml of serum of the test tube marked as Test (T) only and mixed well. The

contents are mixed well and then it is incubated at 37°C for 15 min. Then add 1ml of Folin’s

phenol reagent to all the test tubes followed 0.1 ml of serum is added to the test tube marked as

Blank (B) only. The contents are mixed well and then all the test tubes are incubated at 37°C for

15 min. Then the contents are centrifuged, 4 ml of supernatant is taken and add 1 ml of 15 %

sodium carbonate to all the test tubes. The contents are mixed 11`well and allowed to stand for

15 min. the absorbance is measure the OD at 640nm using red filter.

NORMAL VALUES OF SGPT:-

SERUM - 22 – 92 IU/L

RESULT:-

Activity of alkaline phosphatase = ___________ IU /L

Enzyme assays Salivary Amylase, SGOT, SGPT etc

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Transcript of Enzyme assays Salivary Amylase, SGOT, SGPT etc