week 3 purine practical

Instructor Information:
- Name: Dr. Gudrun Stenbeck
- Location: Heinz Wolff building, room 231
- Contact Phone: 265891
- Email: gudrun.stenbeck@brunel.ac.uk

ADA: Adenosine Deaminase
PNP: Purine Nucleoside Phosphorylase
Guanine Deaminase: An enzyme involved in purine metabolism.
Purine Degradation Leads to Uric Acid Formation: The metabolism of purines results in the production of uric acid, an important biological compound.

Hyperuricemia: An elevated level of uric acid in the blood.
- Consequence: Can lead to precipitation of urate crystals in the joints, resulting in gout.
Gout:
- Prevalence: Ranges from <1% to 6.8% in the general population.
- Incidence: Estimated between 0.58 to 2.89 per 1,000 person-years.
- Demographics: More prevalent in men than women; associated with increasing age and certain ethnic groups.
Risk Factors for Gout:
- Obesity
- Dietary factors
- Comorbid conditions including cardiovascular disease and chronic kidney disease.
Comorbidities Associated with Gout:
- Increased risk of cardiovascular disease
- Chronic kidney disease
- Obstructive sleep apnea
- Osteoporosis
- Venous thromboembolism
Primary Treatments for Gout:
- Dietary changes
- Analgesics
- Steroids
- Allopurinol and febuxostat (medications to lower uric acid levels)

Ischaemia-Reperfusion Injury:
- Definition: Tissue and cellular damage that occurs due to inadequate blood flow followed by the restoration of blood supply.
- Consequences of Ischaemia:
- Lack of oxygen availability
- Depletion of high-energy molecules like ATP
- Accumulation of toxic metabolites.
Xanthine Dehydrogenase to Xanthine Oxidase Conversion:
- Description: Reduced blood supply results in the enzyme xanthine dehydrogenase converting to xanthine oxidase, which catalyzes the production of superoxide ( $O_2^-$ ) instead of $NADH$ .
Reactive Oxygen Species (ROS):
- Onset of reperfusion leads to increased production of ROS, contributing to tissue damage.

Biochemistry Text Reference:
- Reference to the book "Biochemistry, 5th Ed; W.H. Freeman" for theoretical background.
Practical Measurement Protocol:
- Measure at Wavelengths:
- 300 nm
- 550 nm in the presence of cytochrome C

Experimental Conditions:
- Measure the rates of enzyme reactions before and after added substrates or inhibitors.
Experimental Setup Details:
- Experiments:
- Experiment 1:
  - Buffer: Xanthine
  - Total Time: 3 minutes
  - Addition: None
- Experiment 2:
  - Buffer: Xanthine
  - Total Time: 3 minutes
  - Addition: SOD (Superoxide Dismutase, 10 mL)
- Experiment 3:
  - Buffer: Xanthine
  - Total Time: 3 minutes
  - Addition: Allopurinol (20 mL)
- Experiment 4:
  - Buffer: Hypoxanthine
  - Total Time: 3 minutes
  - Addition: None
- Experiment 5:
  - Buffer: Hypoxanthine
  - Total Time: 3 minutes
  - Addition: SOD (10 mL)
- Experiment 6:
  - Buffer: Hypoxanthine
  - Total Time: 3 minutes
  - Addition: Allopurinol (20 mL)
Timing for Measurements:
- 1 minute and 1.5 minutes intervals for measurements.

For Experiments 1-6:
- Measure reduction of cytochrome C at wavelength 550 nm.
For Experiments 7-12:
- Measure uric acid at wavelength 300 nm.

HAZARDS:
- Some reagents are toxic and irritants (e.g., Allopurinol).
Safety Precautions in the Lab:
- No eating or drinking in the lab; wait until outside.
- Wear lab coats and safety goggles.
- Hair must be tied back.
- Check waste streams for proper disposal methods.

Clear up the following items:
- Xanthine Buffer, Hypoxanthine Buffer: Left on the side of the bench for collection.
- Cytochrome C: Dispose of in Bio Bin.
- Xanthine Oxidase (XOD): Dispose of in Bio Bin.
- Superoxide Dismutase (SOD): Dispose of in Bio Bin.
- Allopurinol: Dispose of in Bio Bin.
- Liquid in Used Cuvettes: Empty into LIQUID WASTE beaker.
- Used Cuvettes: Dispose of in Bio Bin.
- Used Parafilm: Dispose of in Bio Bin.
- All other items: To be left on the bench in an organized manner.