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8 Glutamate Dehydrogenase (Nash and Davies 1975)

8 Glutamate Dehydrogenase (Nash and Davies 1975)

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11.10



Malate Dehydrogenase



231



Conduct the electrophoretic run as described. After electrophoresis incubate the

gels in a solution containing 8 mM monosodium glutamate, 0.2 mM methylthiazol

tetrazolium (MTT) or nitrobluetetrazolium (NBT), 0.1 mM phenazine methosulphate, 0.2 mM NAD, 1.2 mM sodium cyanide in 200 mM Tris-HCI buffer

pH 7.5. The staining solution for glutamate dehydrogenase (NADP-dependant)

contains 0.2 M NADP instead of NAD.



11.9



Indolacetic Acid Oxidase



Enzyme extract: As prepared for the assay of the enzyme.

Incubation of the gel: Incubate the gel in staining mixture of 1 mg potassium

indoleacetate, 0.08 mg 2,3,6-trichlorophenol and 2 mg Fast blue BB per ml in

60 mM phosphate buffer (pH 6.0) at 30 C for sufficient time (preferably overnight).



11.10



Malate Dehydrogenase (Honold et al. 1966)



Enzyme extract: Prepare the extract as described for the assay of the enzyme.

Staining of the gel after electrophoresis: First pre-incubate the gels for

15–20 min in 200 mM Tris-HCl (pH 7.5) buffer. Then transfer the gel to a solution

containing 16 mM L-malate, 0.2 mM NAD, 0.25 mM methylthiazol tetrazolium,

0.8 mM phenazine methosulfate, 4 mM MgCI2 and 1.2 mM sodium cyanide in

200 mM Tris-HCl (pH 7.5) buffer. To stain MDH (NADP-dependant), use NADP

instead of NAD.



Chapter 12



Chromatographic Separations



12.1



12.1.1



Separation and Identification of Amino Acids

by Descending Paper Chromatography

Principle



Amino acids in a given mixture are separated on the basis of differences in their

solubilities and hence differential partitioning coefficients in a binary solvent

system. The amino acids with higher solubilities in stationary phase move slowly

as compared to those with higher solubilities in the mobile phase. The separated

amino acids are detected by spraying the air dried chromatogram with ninhydrin

reagent. All amino acids give purple or bluish purple colour on reaction

with ninhydrin except proline and hydroxyproline, which give a yellow coloured

product. The reactions leading to the formation of purple complexes are given

below (Fig. 12.1).



12.1.2

1.

2.

3.

4.

5.

6.

7.



Reagents



Whatman No. 1 filter paper sheet.

Micropipette.

Oven (105 C)

Drier

Sprayer

Chromatographic chamber saturated with water vapours.

Developing solvent: Butanol, acetic acid and water in the ratio of 4:1:5 in

a separting funnel and mix it thoroughly. Allow the phases to separate out

completely. Use the lower aqueous phase for saturating the chamber. The upper

organic phase is used as mobile phase.



R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,

DOI 10.1007/978-1-4419-9785-2_12, # Springer Science+Business Media, LLC 2011



233



234



12



Chromatographic Separations



O



O

R

OH

OH



COOH



O

Ninhydrin

O



Amino Acid



O

Hydrindantrin



O



O



O



H

+ RCHO + NH3 + CH2

OH



+ H 2N C H



H H

OH

+H N

+

OH

H HO



Ninhydrin



O



O

C=N C

O



O



Ammonia



Hydrindantin



+ 3H2O



O



Purple coloured product



Fig. 12.1 Reaction for ninhydrin test



8. Ninhydrin spray reagent: Prepare fresh by dissolving 0.2 g ninhydrin in 100 ml

acetone.

9. Standard amino acids: Prepare solutions of authentic samples of amino acids

such as methionine, tryptophan, alanine, glycine, threonine, etc. (1 mg/ml of

10% iso-propanol).

10. A sample containing mixture of unknown amino acids.



12.1.3



Procedure



1. Take Whatman No. 1 filter paper and lay it on a rough filter paper. Throughout

the experiment care should be taken not to handle the filter paper with naked

hands and for this purpose either gloves should be used or it should be handled

with the help of folded piece of rough filter paper.

2. Fold the Whatman No. 1 filter paper about 2.0–2 cm from one edge. Reverse

the paper and again fold it 2 cm further down from the first fold.

3. Draw a line across the filter paper with a lead pencil at a distance of about 2 cm

from the second fold. Put circular marks along this line at a distance of 2.5 cm

from each other.

4. With the help of a micropipette or micro-syringe apply 20 ml of solution of each

standard amino acid on a separate mark. Also apply spot of the sample or

mixture to be analyzed, preferably on the mark at centre of this base line. The

size of the spot should be as small as possible so that the developed spots are

compact and do not overlap.

5. Hang the filter paper in a chromatographic chamber which has previously been

saturated with aqueous phase of the solvent system. This is done by keeping

Petri plates containing the aqueous phase at the bottom of the chamber. The

paper is hung from the trough/tray and a glass rod is kept to hold it in place.

Care should be taken to ensure that the base line should not get submerged



12.1



6.

7.



8.

9.

10.

11.

12.



Separation and Identification of Amino Acids by Descending Paper . . .



235



when the mobile phase is added to the trough otherwise the spotted material

would get dissolved in the solvent.

Close the chamber firmly so that it is airtight. Allow sufficient time for

cellulose fibres of the paper to get fully hydrated.

Pour the mobile phase through the holes provided on the lid of the chamber into

the trough. Replace the rubber bungs in the hole and allow the mobile phase to run

down the paper till the solvent front reaches about 5 cm from the opposite edge.

Remove the paper and mark the solvent front with lead pencil and let it dry at

room temperature.

Spray the filter paper (chromatogram) with ninhydrin reagent and after drying it

at room temperature, transfer it to an oven at 105 C for 5–10 min.

Blue or purple coloured spots would appear on the paper. Mark the boundary of

each spot with lead pencil.

Measure the distance between the centre of the spots and also the distance of

the solvent front from the base line.

Calculate the Rf value of standard amino acids as well as those in the given

mixture or sample.

Rf ¼



Distance traveled by unknown amino acid

:

Distance traveled by the solvent system



13. Identify the amino acids in the mixture or sample by comparing their Rf values

with those of the reference standards.



12.1.4



Observations and Calculations



Distance travelled by the solvent front from base line ¼ x cm

Distance travelled by glycine from base line ¼ a cm

Distance travelled by alanine from base line ¼ b cm

Distance travelled by threonine from base line ¼ c cm

Distance travelled by methionine from base line ¼ d cm

Distance travelled by spot no.1 in sample from base line ¼ a cm

The sample contains glycine since Rf value of spot no. 1 is identical to that of

glycine standard.



12.1.5



Note



• The chromatography should be carried out in temperature controlled room

because any fluctuation in the temperature would cause the uneven flow of the

solvent and may alter the Rf value.



236



12



Chromatographic Separations



• The paper should not be touched with naked hands because sweat on hands

contains significant amount of amino acids.

• The spots of the applied sample should be as compact as possible. Larger the

spot, poorer will be the resolution.

• At the time of fixing paper in chromatography chamber, it should be ensured that

the base line on which the sample has been applied does not dip into the solvent

otherwise the sample might get washed away in the solvent.

• Allow sufficient time for filter paper to absorb sufficient water (which will act as

a stationary phase) before pouring the solvent into the tank. Inadequate conditioning or equilibration will result in improper or poor quality resolution.

• Solvent front should advance in a straight line and should not be zig-zag or

sloping but should be parallel to the base line.

• Dry the paper thoroughly before spraying with the detection reagent. Wet paper

may interfere with the appearance of evenly shaped compact spots.



12.2



12.2.1



Separation and Identification of Amino Acids

by Ascending Paper Chromatography

Reagents and Materials



Same as given in above experiment except that cylindrical chromatography

chambers are needed for this experiment.



12.2.2



Procedure



1. Take Whatman No. 1 filter paper sheet of appropriate size so that it can

be rolled into a cylinder and can be accommodated in the cylindrical

chromatographic jar.

2. Draw a base line 2 cm from one of the breadthwise edge of the paper. Put small

circular marks along the base line in such a way that the distance from the edge

of the paper and the first spot and the distance between the adjacent spots is not

less than 2.5 cm.

3. Apply 20 ml aliquots of the standard amino acids and of the sample on different

spots. Diameter of the spotted material should be as small as possible and, if

required, the applied solution may be dried prior to loading additional volume.

4. Roll the paper into a cylinder; fasten its edges with a paper clip. Pour sufficient

volume of the mobile phase into the chromatographic jar which has been earlier

saturated with water vapours by lining the tank with filter paper saturated with

aqueous phase of the solvent system.



12.3



Separation and Identification of the Amino Acids by Two-Dimensional. . .



237



5. Gently place the rolled filter paper upright in the jar ensuring that it does not

touch the sides of the chamber and at the same time taking care that the base

line where the spots have been applied does not dip into the solvent.

6. Close the tank with n airtight lid or a glass plate to which sufficient amount of

silicon grease has been applied.

7. Leave the set up undisturbed and allow the solvent to move up fill it reaches

about 5 cm from the upper edge.

8. Remove the chromatogram from the chamber and air dry it.

9. Spray the paper with ninhydrin reagent and let it dry again at room temperature

prior to transferring it to an oven at 105 C for 5–10 min. Locate the position of

amino acids from the bluish or purple coloured spots on the chromatogram.

10. Calculate the Rf values of the standard amino acids and those is the sample or

mixture as described in above experiment.

11. Identify the amino acids in the mixture or sample by comparing Rf values with

those of applied standard amino acids.



12.3



12.3.1



Separation and Identification of the Amino Acids

by Two-Dimensional Paper Chromatography

Principle



Amino acids having very close Rf values in a particular solvent system may appear

as a single or overlapping spots in a single dimensional chromatography and may be

mistaken as one component. They can be separated into individual components by

developing the chromatogram again in a direction perpendicular to the first run in a

second solvent system in which they have different Rf values. The main limitation

of this method is that only one spot either of the sample or of a standard amino acid

can be applied on each filter paper sheet necessitating running of a large number of

chromatograms for the standard amino acids.



12.3.2



Reagents



1. As in previous experiment with an additional chromatographic chamber for the

second solvent system.

2. Solvent system No.2: Phenol: water (80:20, w/v) is used as second solvent

system. Add 125 ml of water to 500 g to phenol. Add a few drops of ammonia

(0.88%) to this mixture just before use (CAUTION-Phenol is corrosive and can

cause burns on the skin).

3. Standard amino acids: Prepare 1% solution of standard amino acids such as

aspartate, glycine, serine, arginine in 10% iso-propanol (u/v).



238



12.3.3



12



Chromatographic Separations



Procedure



1. Lay the chromatographic paper sheet flat on the rough filter paper using gloves.

2. Draw a base line 5 cm from one of the edges of the paper.

3. Draw another line perpendicular to the first line again 5 cm away from the

adjacent edge.

4. Apply 60 ml of the sample solution or given mixture containing unknown amino

acids at the point of intersection of these two lines. The sample should be

applied in small volumes at a time with the help of a micropipette with

intermittent drying to ensure that the zone of applied solution is as small as

possible.

5. Repeat the same procedure for a mixture of three standard amino acids using a

separate chromatographic sheet for each mixture. The composition of mixture

of standard amino acids should be such that each amino acid is present in at

least two different mixtures so that its identity can be established.

6. Hang the paper in the chromatographic tank whose interior has previously been

saturated with aqueous phase of solvent system No.1 (Butanol: Acetic acid:

Water mixture is 4:1:5).

7. After allowing an equilibration period of half an hour, pour the solvent No.1

into the trough of the chamber and let it run till t\it is about 10 cm from the

opposite edge of the paper.

8. Take the paper out, air dry it, turn it at 90 C angle and now develop the paper in

the second chromatographic chamber using the solvent system No.2 (Phenol:

Water).

9. Remove it when the solvent has travelled upto about 10 cm from the

opposite end.

10. Dry it at room temperature and spray it with ninhydrin reagent. After air drying

it, keep the chromatogram in an oven at 105 C for 10 min. Mark the blue and/or

purple coloured zones which appear on the paper.

11. Calculate the Rf value of the standard amino acids and those in given mixture as

given in previous experiment in both the solvents. From these values identify

the amino acids in the given mixture.



12.4

12.4.1



Identification of Lipids by TLC

Principle



Lipids generally exists as lipoprotein complexes and these need to be isolated.

Lipids, being soluble in non-polar organic solvents and proteins being soluble in

polar aqueous solvents, the efficient lipid extraction can be achieved only when an

aqueous solvent like ethanol or methanol is included in the non-polar organic

solvent like chloroform and diethyl ether. This would help in breaking the



12.4



Identification of Lipids by TLC



239



lipoprotein complexes. Extracted lipid components can be separated on TLC base

on their differential mobility along the porous stationary phase such as silica gel and

these can be located by spraying the plates with either 2’,7’-dichlorofluorescein or

50% sulfuric acid.



12.4.2



Reagents and Materials



1.

2.

3.

4.

5.



TLC tank

Oven set at 110 C

Glass plates (20 Â 20 cm) for TLC

Ultraviolet lamp

Solvent system: Petroleum ether (b.p. 60–70 C) or hexane: diethyl ether: glacial

acetic acid (80:20:1, u/v)

6. Lipid standards: Various lipids such as cholesterol acetate, vitamin A palmitate,

triacyl glycerol (e.g., trioleate, tripalmitate, tristearate)

7. Sulfuric acid (50%, u/v)

8. 2’,7’-dichlorofluorescein: Prepare 0.2% solution of 2’,7’-dichlorofluorescein in

95% (u/v) ethanol



12.4.3



Procedure



1. Extraction of lipids from sample: Grind 1 g of the tissue in the extraction solvent

[either ethyl ether: ethanol (3:1) or chloroform: methanol (2:1)] in pestle and

mortar. Transfer the homogenate to a separating funnel. Shake the contents

vigorously and allow it to stand till the two phases have completely separated.

Drain out the lower organic layer which contains the lipids. Evaporate the

solvent under organic layer which contains the lipids. Evaporate the solvent

under vacuum and keep the concentrated lipid extract protected from light under

N2 almosphere.

2. Prepare the TLC plates using Silica Gel G as the adsorbent as described in

previous experiment.

3. Activate the TLC plates at 110 C for 30 min, cool them in a desiccator and spot

the lipid samples, standards as well as unknown sample.

4. Develop the plates in the solvent system consisting of petroleum ether or hexane:

ethyl ether: glacial acetic acid (80:20:1) till the solvent has travelled upto 1 cm

from the opposite side of the plate.

5. Remove the plate and allow it to air dry.

6. Locate the lipid spots by either of the following methods:

(i) Spray the plate with 2’,7’-dichlorofluorescein and examine it under UV light.

Lipids show up as green fluorescent regions against the dark background.



240



12



Chromatographic Separations



(ii) Spray the plate carefully with 50% H2SO4 and heat it in an oven at 110 C

for 10 min. Areas containing lipids gel charred and appear as black spots.

7. Calculate the Rf value of the lipid components in the sample and identify them

by comparing their Rf values with lipid standards.



12.5



12.5.1



Separation of Pigments by Adsorption Column

Chromatography

Principle



Different pigments get adsorbed to alumina to different extents. They can be selectively desorbed by using mobile phase of increasing polarity in a stepwise manner.



12.5.2

1.

2.

3.

4.

5.

6.

7.

8.



Materials and Reagents



Leaves/flowers

Pestle and mortar

Glass column

Whatman No.1 filter paper

Alumina

Benzene: methanol (2:1)

Sodium sulphate (anhydrous)

Acetone



12.5.3



Procedure



(a) Preparation of extract:

1. Homogenize 5 g leaves or flowers in a pestle and mortar, using sand as an

abrasive in 20 ml of benzene: methanol (2:1) adding a small amount of this

extractant at a time.

2. Filter the extract through Whatman No.1 filter paper and transfer the filtrate

to a separating funnel.

3. Add 10 ml of water to the filtrate and after shaking the contents and allowing

the phase to separate out, drain out the lower aqueous methanol layer. Repeat

this step. Avoid very vigorous shaking.

4. Collect the benzene layer in a beaker and add small amount of solid

anhydrous Na2SO4 to remove the traces of moisture.

5. Decant the clear benzene layer to another beaker and concentrate the extract

by evaporating the solvent on a boiling water bath.



12.6



Separation and Identification of Sugars by Adsorption TLC



241



(b) Column preparation:

1. Mount a burette or a glass column vertically on a burette stand with the help

of clamps.

2. Place lightly a plug of glass wool at the base of the burette and close the

stopcock or outlet at the bottom of the column.

3. Take 5 g of alumina (adsorbent) previously dried at 120 C for 8 h and prepare

its slurry in benzene. Pour the slurry carefully into the column or burette, by

gentle tapping of the column so that no air bubbles get trapped in the adsorbent.

4. Allow the adsorbent to settle by opening the outlet. After the adsorbent has

completely settled, add 20 ml of benzene and let it pass out of the column. Care

should be taken not to let the adsorbent to get dried. When about 1 cm layer of

the solvent remains at the top of the chromatographic bed, close the outlet.

Sample application:

1. Allow the solvent at the surface of the column to drain out slowly and

transfer the leaf or flower extract with the help of pipette without disturbing

the surface of the column adsorbent. Let it enter into the column and then

add a few drops of benzene to wash the traces of the extract sticking to the

wall of the column.

2. Add 20 ml more benzene to wash out the column of any unadsorbed material.

(c) Column development:

1. For desorption of the adsorbed substance change the polarity of the solvent in

a stepwise manner. After 20 ml of benzene has passed through the column

add 10 ml of 5% acetone (u/v) in benzene and let it percolate through the

column and collect 1–2 ml fractions of the effluent from the outlet. Continue

increasing the concentration of acetone in benzene at every succeeding step.

Finally pass pure acetone through the column.

• Note the change in the colour of the collected fractions. In case of the leaf

extract, the initial fractions are colourless followed by yellow coloured and

then by the green coloured ones. The colourless fractions do not contain

any pigments but it is quite possible that these fractions may contain some

UV absorbing materials.



12.6



12.6.1



Separation and Identification of Sugars

by Adsorption TLC

Principle



Sugars get separated on the basis of differential adsorption onto silica gel. The

sugars which have higher affinity for stationary phase are adsorbed more strongly



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