Practical B Flashcards
animals do not store …
amino acids
excess of amino acids must be…
deaminated so that the carbon skeletons can be used as metabolic fuels (or converted to glycogen or fats for future use).
other name for keto acids
oxo acids
Transaminases-
(=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.
Transaminases have …. as a tightly bound cofactor
This is derived from ….
pyridoxal phosphate
vitamin B6.
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).
aspartate
glutamate
Oxidative deamination allows the removal of an amino group from …. This reaction is catalyzed by …
glutamate.
glutamate dehydrogenase:
Glutamate dehydrogenase is unusual in that it will use …
either NAD+ or NADP+ as
coenzyme.
The toxic ammonium ion is transported (as …) to the liver where it is converted into …. which enters the urea cycle.
glutamine
carbamoyl phosphate
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?
(this means many changes of the dialysis buffer)
Rozwiń skrót TLC
Thin Layer Chromatography
TLC: Thin layer chromatography is a quick and convenient way of …
separating many types of
compound.
TLC: Separation is effected by … , which may be ….
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).
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.
The 2,4 dinitrophenyl-hydrazones
yellow
spraying with ninhydrin (and heating)
a purple
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?
none visible
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?
glutamate
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?
glutamate + alanine
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?
glutamate
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?
a standard of the 2,4 dinitrophenylhydrazone of pyruvate
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?
DNPH
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?
oxoglutarate + pyruvate
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?
oxoglutarate + DNPH
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?
2,4 dinitrophenylhydrazine (DNPH or Brady’s reagent) control.
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?
wykryto 2-OXOGLUTARATE
k
k
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?
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).
Plate I (amino acids): What happens after tunning the plate in the solvent system I?
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
Plate II (Dinitrophenylhydrazones of oxo-acids): What happens after tunning the plate in the solvent system II?
The following instruction should be followed:
Dry in the fume cupboard after marking solvent front. DO NOT spray this plate.
Plate II (Dinitrophenylhydrazones of oxo-acids): What happens after tunning the plate in the solvent system II?
The following instruction should be followed:
Dry in the fume cupboard after marking solvent front. DO NOT spray this plate.
The unreacted dinitrophenylhydrazine runs irregularly …
near the top of the plate.
Pyruvate DNP often produces ….
2 spots close together (isomers).
