Transamination and deamination Practical PDF

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Transamination and deamination in action • Transaminase - catalyze transamination reaction between an amino group and an α-keto acid. • Deaminase - catalyze the removal of an amino group. • Importance in the breakdown of amino acids in the body. Our body is unable to store excess amino acids so they need to be broken down. If the amino group is released, it would leave free ammonia which is toxic. So instead the amino group is converted into nontoxic urea in the liver, released into blood where it travels to the kidneys then excreted by the urine.

• Importance in fed state and starved state. In fed state: the excess amino acids will be metabolized into basic carbon skeleton, particularly often Kreb cycle intermediates, so they can be fed into catabolic pathway and broken down to energy. In starved state: muscle tissue break down to release amino acids that can be transaminated to alanine. The alanine passes to the liver and in liver is then converted into pyruvate.

Our experimental aims • To understand the basic biochemical principle behind transamination and deamination. • To understand the importance and use of controls in experiments. • To be able to use TLC to detect the presence of amino acids and oxo acids accurately using appropriate controls. • To be able to interpret TLC plates from any given data. • To understand the nature and use dialysis. Transamination reaction

Enzymatic reaction so reactants will be observed on TLC plate.

Deamination reaction

Transaminase and deaminase enzymes • Purified from lambs hearts (a source of • Macerate the tissue • Resuspend in buffer • Centrifuge to remove cell debris’s • Soluble enzymes in the supernatant

muscle tissue)

Dialysis To remove any small soluble molecules like amino acids that are present in the extract. The solution is put into these bags with semi-permeable membrane. The pores are smaller than the enzymes which means small molecules like amino acids and cofactors will diffuse out of the bag while enzymes are trapped within. The samples are placed in iced, cold buffer. The buffer is changed several times so the concentration of small molecules in extract can be reduced. As in later stage, we want to add back in the amino acids and oxo acids as reactants. This is to prevent contamination from these small molecules.

Laboratory processes • Add various combinations of amino and 2-oxo acids (see table in lab video and on final worksheet) • Add heart extract in buffer to all samples • Incubate samples at 37℃ • Separate the amino and oxo acids using TLC • Different detection techniques for amino and oxo acids

Detection of reagents - amino acids • Ninhydrin spray (on TLC after the separation) • Detects ammonia or amines • When reacts forms a deep purple color Detection of reagents - 2-oxo acids • 2,4-dinitrophenylhydrazine (DNPH) • Reacts with aldehydes and ketones • Forms a yellow derivatives • Need additional incubation steps to create the yellow derivatives Excess DNPH is adde. The unreacted chemical sometimes could be seen on the TLC plate at point near the solvent front.

Analysis of TLC plates • Identify controls • Use controls to work out what is present in reaction tubes • What can you see in tube 10? There are two isomers of pyruvate so they perform slightly different on the plate....