Flashcards
Ebio Labs 4 Biochem
Cards in this deck (37)
- 1What reaction does Pyruvate Kinase catalyse?Phosphoenolpyruvate + ADP > ATP + Pyruvate
- 2What is the result of RBCs having no mitochondria?They are particularly reliant on glycolysis as the main metabolic pathway for ATP generation (glycolysis happens in the cytoplasm).
- 3What is an important allosteric effector of haemoglobin produced from 1,3-bisphosphoglycerate?2,3-bisphosphoglycerate.
- 4If the iron atom in haemoglobin undergoes oxidative stress, what takes place? What does our body do about this to limit the damaging effects?The iron atom can be oxidised from the ferrous (Fe2+) state to the ferric (Fe3+) state to form methaemoglobin - this state cannot bind oxygen. We therefore reduce this to be able to bind oxygen.
- 5What is the result of oxidative damage to the polypeptide chain in haemoglobin and to lipid membranes of RBCs?- Alter haemoglobin's protein structure so that it precipitates inside the cell forming Heinz bodies.
- Leads to membrane fragility and red blood cell lysis. - 6What is a term used to describe the oxidative damage induced on RBC lipid membranes?Peroxidation.
- 7What is Glutathione (reduced form GSH)?A small tripeptide (glycine-cysteine-glutamate) which plays a key role in combating oxidative stress in RBCs.
- 8Describe how Glutathione combats Oxidative Stress.- Glutathione reductase enzyme uses electrons/ protons from NADPH + H+ products of the Pentose Phosphate pathway to reduce Glutathione into GSH.
- In doing this, we supply reduced GSH to gliutathione peroxidase which uses GSH to breakdown reaction oxygen species / ROS such as hydrogen peroxide into water.
- This prevents Hb oxidation of iron group, thus prevents protein damage.
- This prevents lipid oxidation. - 9When might a Glutathione shortage occur in the body?The synthesis of Glutathione requires ATP, and therefore, any defects which affect the ability of RBCs to produce ATP will decrease the amount of GSH available and hence increase the potential for oxidative damage.
- 10List some key symptoms of people suffering from PK deficiency.- Lethargy
- Anaemia (constant or episodic) - 11Why can PK deficiency be difficult to diagnose?- Carrier are generally asymptomatic
- Full PK deficiency can be asymptomatic. - 12What do the PK genes of the majority of people with PK defects appear like?They have a single autosomal recessive mutation in the PK gene.
- 13What do the PK genes of wild type people appear like?2 x normal copies of the PK gene with full PK activity in their RBCs.
- 14What do the PK genes of the minority of people with PK deficiency appear like?2 x copies of the recessive mutant allele with undetectable levels of PK activity in their RBCs.
- 15What are restrictive enzymes?They cut double stranded DNA at specific sequences.
- 16How does the restriction enzyme Hinfl (which recognises GACTC of the PK gene) behave on PCR fragments from normal, wild-type people?It cuts GACTC into 2 smaller fragments.
- 17How does the restriction enzyme Hinfl (which recognises GACTC of the PK gene) behave on PCR fragments from people who are previously shown to have a mutation which alters the Hinfl site sequence to AACTC?It is not cut by the enzyme, so remains as one fragment.
- 18Briefly describe DNA electrophoresis.- DNA loaded onto agarose gel.
- Current passed through the gel.
- DNA is uniformly negatively charged.
- Molecules migrate towards the +ve electrode/ anode.
- Smaller molecules are less impeded by the agarose gel and travel faster than larger ones.
- A DNA standard may be loaded onto the gel for comparison.
- The gel contains a DNA binding dye that fluoresces under UV light, allowing visualisation of fragments under UV for analysis. - 19How many bands on the DNA electrophoresis would you expect to see in a sample from someone who is homozygous for the healthy gene (+/+)?2
- 20How many bands on the DNA electrophoresis would you expect to see in a sample from someone who is homozygous for the mutant gene (-/-)?1
- 21How many bands on the DNA electrophoresis would you expect to see in a sample from someone who is heterozygous for the healthy/ mutant gene (+/-)? Why?3 - The enzyme will cleave the PCR product of the healthy gene into two fragments of unequal length. It will leave the mutant gene as a single (larger) fragment, so in total = 3 bands.
- 22In order to measure the rate of reaction (of PK activity) we need to detect the levels of at least one of the molecules involved in the reaction. None of the molecules in the main PK reagent can be detected using a spectrphotometer... what molecule can we link in and how?NADH - We can measure NADH utilisation by Lactate dehydrogenase in order to convert pyruvate into lactate.
NADH measures light at 340nm; but the product of the reaction does not.
So, the decrease in absorbance at 340nm can be used as a surrogate for PK activity. - 23Over the course of your PK assay, you measure a decrease in absorbance of NADH from 0.84 to 0.51 over the course of 20 seconds. What is the decrease in [NADH] during that time?0.84 - 0.51 = 0.33
A = ecl
0.33 = 6220 x c x 1
0.33 / 6220 = 5.3055e-5mol.L^-1
= 53micromolesL^-1 - 24What is the NADH concentration of a solution which gives an absorbance at 340nm of 0.59? (to 2 sig fig) e = 6220L.mol^-1.cm^-1 Pathlength =1cmA = ecl
0.59 = 6220 x c x 1 =
9.5e-5 mol.L^-1. =
95micromol.L^-1 - 25Briefly describe how the SDS-PAGE can be used in PK deficiency.- Analytical tool.
- Used to separate proteins on the basis of their size.
- By comparing the protein separation pattern of healthy PK to mutant PK, we may be able to understand more about the effect of the mutation of the PK gene on the PK gene product/ the protein. - 26You carry out the PK assay described earlier and detect an absorbance change of 0.14 in one minute. What is the concentration change of NADH during this time? What is the concentration change of PEP during this time? (2 sig fig)A = ecl
0.14 = 6220 x c x 1
0.14 / 6220 = 2.251e-5 mol.L^-1
C = 23micromoles.L^-1 of both NADH and PEP due to the 1:1 ratio. - 27What are the main differences between Agarose gel electrophoresis and SDS-PAGE?Agarose gel - separates DNA according to size.
SDS-PAGE - separates proteins according to size. - 28You obtain an average absorbance change per minute value of 0.363. Calculate the [NADH] change per minute for Liz's sample in µmol.L-1.min-1 to one decimal place.A = ecl
0.363 = 6220 x c x 1
0.363 / 6220 = 5.836e-5mol.L^-1
= 58.4micromoles.L^-1 - 29What is the NADH concentration of a solution which gives an absorbance at 340 nm of 0.76? (micromoles)A = ecl
0.76 = 6220 x c x 1
0.76 / 6220 = 1.223e-4mol.L^-1
122micromoles.L^-1. - 30If the change in [NADH] per minute for Hannah's blood sample is 54 µmol.min-1.L-1, what is the [PEP] per minute in the same units?54 (1:1 ratio).
- 31The coding region of the PK gene consists of 1725 base pairs. How many amino acids does the protein contain?1725 / 3 = 575
- 32If a disease is caused by an autosomal dominant mutation, what percentage of offspring produced by two heterozygote individuals are likely to be affected by the disease?75%
- 33Which is more accurate, estimating values of Km and Vmax from the Michaelis-Menten plot or from the Lineweaver-Burk plot?Lineweaver-Burk plot
- 34Approximately what band size(s) on the SDS-PAGE gel could be seen for heterozygous individuals of the PK mutant gene?- 60 kDa (healthy copy) and 25 kDa (mutated copy).
- 35From my experimental results, what effect did the PK mutation have on the PK gene enzyme?From the SDS-PAGE gel, the mutant protein is significantly smaller than the functional one.
The size difference strongly implies some form of truncation mutation.
This must be significant enough to produce a non-functional enzyme, as seen from the PK assay results. - 36What is the IU / International Units?Often PK activity in blood samples is expressed in IUs. The IU is defined as the micromoles of pyruvate produced per minute per mg of RBC protein (micromoles.min^-1.mg^-1).
- 37If you measured PK activity in a reaction with a total volume of 1 mL in a cuvette containing 0.5 mg of red blood cell protein and obtained a rate of NADH disappearance of 200 µmoles.L-1.min-1, what is the activity of PK expressed in IU?200micromoles.L^-1.min^-1...
= 0.2micromoles.mL^-1.min^-1.
If 1mL contains 0.5mg protein.
Then in 1mg...
= 0.4micromoles.mg^-1.min^-1.