Practical B

Question 1

animals do not store …

Answer 1

amino acids

Question 2

excess of amino acids must be…

Answer 2

deaminated so that the carbon skeletons can be used as metabolic fuels (or converted to glycogen or fats for future use).

Question 3

other name for keto acids

Answer 3

oxo acids

Question 4

Transaminases-

Answer 4

(=amino transferases) are enzymes which transfer an amino group from an α-amino acid to a 2-oxo acid (same as an α-keto acid) producing a new amino acid and a new 2-oxo acid.

Keq. is close to 1 for transaminases.

Question 5

Transaminases have …. as a tightly bound cofactor

This is derived from ….

Answer 5

pyridoxal phosphate

vitamin B6.

Question 6

Transaminases allow
interconversion of most amino acids and the “funnelling” of amino groups to …
(used in urea cycle) or to … (which can then undergo oxidative deamination).

Answer 6

aspartate

glutamate

Question 7

Oxidative deamination allows the removal of an amino group from …. This reaction is catalyzed by …

Answer 7

glutamate.

glutamate dehydrogenase:

Question 8

Glutamate dehydrogenase is unusual in that it will use …

Answer 8

either NAD+ or NADP+ as

coenzyme.

Question 9

The toxic ammonium ion is transported (as …) to the liver where it is converted into …. which enters the urea cycle.

Answer 9

glutamine

carbamoyl phosphate

Question 10

From the sentence below:

The heart is minced, homogenised in ice-cold buffer and centrifuged at 20,000g x 20
minutes. The supernatant is then dialysed exhaustively (…) against ice-cold buffer (0.05mol L-1 phosphate, pH 7.4).

What does dialysed exhaustively mean?

Answer 10

(this means many changes of the dialysis buffer)

Question 11

Rozwiń skrót TLC

Answer 11

Thin Layer Chromatography

Question 12

TLC: Thin layer chromatography is a quick and convenient way of …

Answer 12

separating many types of

compound.

Question 13

TLC: Separation is effected by … , which may be ….

Answer 13

allowing a solvent mixture to flow up (by capillary action) a thin layer of very fine particles, …

silica gel, aluminium oxide or cellulose (among other media).

Question 14

TLC: The separated compounds must be visualized.

…. of the oxoacids are …. and can be seen directly.

The amino acids are detected by
…. , which gives …. colour when amino groups are present.

Answer 14

The 2,4 dinitrophenyl-hydrazones

yellow

spraying with ninhydrin (and heating)

a purple

Question 15

Tube 1. 
0.5 ml of: 
0.1 mole L^-1 Na PYRUVATE
0.05 mole L-1 Pi buffer, pH 7.4
"Extract" (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
Add 1.5 ml ethanol

Co zostanie wykryte?

Answer 15

none visible

Question 16

Tube 2. 
0.5 ml of: 
0.1 mole L^-1 Na GLUTAMATE
0.05 mole L-1 Pi buffer, pH 7.4
"Extract" (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml ethanol

Co zostanie wykryte?

Answer 16

glutamate

Question 17

Tube 3.
0.5 ml of: 
0.1 mole L^-1 Na PYRUVATE
0.1 mole L^-1 Na GLUTAMATE
"Extract" (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml ethanol

Co zostanie wykryte?

Answer 17

glutamate + alanine

Question 18

Tube 4. 
0.5 ml of: 
0.1 mole L-1 Na GLUTAMATE
0.005 mole L-1 NAD+
"Extract" (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
Add 1.5 ml ethanol

Co zostanie wykryte?

Answer 18

glutamate

Question 19

Tube 5.
0.5 ml of:
0.1 mole L^-1 Na PYRUVATE
0.05 mole L-1 Pi buffer, pH 7.4
“Extract” (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml 2,4 dinitrophenylhydrazine solution

Co zostanie wykryte?

Answer 19

a standard of the 2,4 dinitrophenylhydrazone of pyruvate

Question 20

Tube 6.
0.5 ml of:
0.1 mole L^-1 Na GLUTAMATE
0.05 mole L-1 Pi buffer, pH 7.4
“Extract” (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml 2,4 dinitrophenylhydrazine solution

Co zostanie wykryte?

Answer 20

DNPH

Question 21

Tube 7.
0.5 ml of:
0.1 mole L^-1 Na PYRUVATE
0.1 mole L^-1 Na GLUTAMATE
“Extract” (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml 2,4 dinitrophenylhydrazine solution

Co zostanie wykryte?

Answer 21

oxoglutarate + pyruvate

Question 22

Tube 8.
0.5 ml of:
0.1 mole L-1 Na GLUTAMATE
0.005 mole L-1 NAD+
“Extract” (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml 2,4 dinitrophenylhydrazine solution

Co zostanie wykryte?

Answer 22

oxoglutarate + DNPH

Question 23

Tube 10.
0.5 ml of:
2X 0.05 mole L-1 Pi buffer, pH 7.4
“Extract” (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml 2,4 dinitrophenylhydrazine solution

Co zostanie wykryte?

Answer 23

2,4 dinitrophenylhydrazine (DNPH or Brady’s reagent) control.

Question 24

Tube 9.
0.5 ml of:
0.1 mole L-1 2-OXOGLUTARATE
0.05 mole L-1 Pi buffer, pH 7.4
“Extract” (in buffer)
After incubation (for 30 min at 37 Celcius) dodano:
1.5 ml 2,4 dinitrophenylhydrazine solution

Co zostanie wykryte?

Answer 24

wykryto 2-OXOGLUTARATE

Question 25

k

Answer 25

k

Question 26

Why, after incubation and making appropriate additions tubes 5-10 are placed back in the 37°C
water bath for 5 minutes and sth is added? What is added?

Why all ten tubes are centrifuged afterwards?

Answer 26

0.5 ml ethyl acetate is added to these tubes.
This extracts the 2,4 dinitrophenylhydrazones.

Centrifuge all ten tubes to remove the precipitated protein and to separate the ethyl acetate (5 - 10).

Question 27

Plate I (amino acids):
What happens after tunning the plate in the solvent system I?

Answer 27

The following instruction should be followed:

Dry with a hair dryer after marking solvent front. Allow to cool, spray with ninhydrin (in the fume cupboard) and heat with a hair dryer until spots become visible

Question 28

Plate II (Dinitrophenylhydrazones of oxo-acids):
What happens after tunning the plate in the solvent system II?

Answer 28

The following instruction should be followed:

Dry in the fume cupboard after marking solvent front. DO NOT spray this plate.

Question 29

Plate II (Dinitrophenylhydrazones of oxo-acids):
What happens after tunning the plate in the solvent system II?

Answer 29

The following instruction should be followed:

Dry in the fume cupboard after marking solvent front. DO NOT spray this plate.

Question 30

The unreacted dinitrophenylhydrazine runs irregularly …

Answer 30

near the top of the plate.

Question 31

Pyruvate DNP often produces ….

Answer 31

2 spots close together (isomers).