Lecture 8: Enzymes for Therapeutic and Diagnostic Applications

Question 1

What are the main general clinical applications of enzymes?

Answer 1

1. As therapeutics
2. As key components in diagnostic/analytical assays

Question 2

What are the concerns involving enzyme Replacement Therapy?

Answer 2

1. Delivery to site of action → very poor oral bioavailability, so need to use invasive methods to administer the drug.
2. Effectiveness of chronic enzyme replacement therapy may be reduced over time.
3. Protein orthologue purified from an animal source may elicit immune response, nut the effectiveness has improved with recombinant enzymes
4. Enzymes are easily degraded so they have a small half life.

Question 3

What happens in Severe combined immunodeficiency disease (SCID)?

Answer 3

Impaired T and B cell function at birth

Question 4

What are the 2 causes that lead to severe combined immunodeficiency disease (SCID)?

Answer 4

1. Most common type: X-linked SCID; mutations in IL-2 receptor gamma subunit
2. Second most common form (15% of SCID): adenosine deaminase (ADA) deficiency.

Question 5

How does adenosine deaminase deficiency lead to severe combined immunodeficiency disease (SCID)?

Answer 5

1. The deficiency stops the breakdown of adenosine and 2-deoxyadenosine
2. Adenosine gets converted to dATP, so adenosine, dATP and 2-deoxyadenosine build up in the body
3. 2 - deoxyadenosine is a cytotoxic metabolite and is generally considered the primary cause of lymphotoxicity.
4. Elevated adenosine contributes to apoptosis and blocks the differentiation of thymocytes, causing severe T lymphopenia.
5. The buildup of dATP, which induces the cell to make cytochrome c, destroys and signals for apoptosis in all rapidly proliferating cells. This includes cells in the GI tract, immune system lymphocytes, and sperm cells.

Question 6

What is the drug used to treat severe combined immunodeficiency disease (SCID) and what is it

Answer 6

Adagen
It’s pegylated bovine adenosine deaminase (ADA)

Question 7

What is the function of PEGylating drugs?

Answer 7

1. PEG chains increase drug’s size, which prevents it from renal clearance and increases circulation half-life
2. Increasing PEG linker length allows improved solubility and decreases accessibility for proteolytic enzymes and antibodies
3. reduce degradation by proteases, lower binding by host antibodies and reduce immunogenicity.

Question 8

What is the main side effect associated with the long term use of PEG-ADA?

Answer 8

The development of anti-ADA antibody which makes the drug ineffective.

Question 9

What gene mutation causes cystic fibrosis?

Answer 9

1. Various mutations a gene encoding for cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel.
2. CFTR regulates components of sweat, digestive juices, and mucus.

Question 10

What is the treatment given to patients with cystic fibrosis?

Answer 10

1. Pulmozyme
2. It is a recombinant human DNase I enzyme (produced in CHO cells) that selectively cleaves DNA and reduces viscosity of mucus.
3. It is administered daily

Question 11

What happens in Gaucher’s disease?

Answer 11

1. It is caused by a deficiency in glucocerebrosidase enzyme (esp beta-glucosidase) that breaks down glycolipids called glucocerebroside
2. Glucocerebroside builds up in the liver and spleen, causing enlargement and affecting their functions

Question 12

What is the drug used to treat Gaucher’s disease and how does it work?

Answer 12

1. Cerezyme
2. It has imiglucerase which is modified placental glucocerebrosidase
3. The glycosylation sites in imiglucerase terminate in mannose which allows them to enter macrophages and digest the glucoerebroside

Question 13

What are the most commonly employed biological samples used in in vitro diagnostics?

Answer 13

1. Enzymes: to detect and/or quantify various medically significant biomolecules or metabolites
2. Antibodies: to detect
   and/or quantify specific substances (antigens).

Question 14

How should the Kcat and Km be for an enzyme used in diagnostic applications?

Answer 14

1. High Kcat
2. Low Km

Question 15

What are the different diagnostic tests for diabetes?

Answer 15

1. Urine glucose
2. Blood glucose
3. HbA1c testing

Question 16

What do the types of urine diagnostic tools used in diabetes test for?

Answer 16

1. Testing for the presence of glucose in urine
2. Testing for the presence of ketones in urine
3. Testing for the presence of proteins and albumin in the urine

Question 17

In an enzyme based assay, what are the two biochemical methods used to make measurements?

Answer 17

1. End point method: let the reaction proceed until no further conversion, and measure total concentration change from beginning to end.
2. Kinetic method: measures the rate at which substrate is consumed or the rate at which
   product is generated at various substrate concentrations

Question 18

What are two commonly used enzymes in assays?

Answer 18

1. Dehydrogenases
   Oxidases

Question 19

What do Clinitest tablets and Diastix strips measure?

Answer 19

Amount of glucose in urine

Question 20

What do Clinitest tablets contain to measure the amount of glucose in the urine and how does it work?

Answer 20

1. Anhydrous benedicts reagent
2. Glucose reduces the Cu2+ in benedict’s reagent to form Cu2O which is coloured and can be detected

Question 21

How do diastix tablets work to test for glucose in urine?

Answer 21

1. Glucose oxidase catalyses oxidation of glucose in the urine to form gluconic acid and h2o2
2. Peroxidase catalyses the oxidation of a potassium iodide chromogen by h2o2, and the chromogen gets oxidised to colours ranging from green to brown which are detected

Question 22

What do photometers and biosensors measure to determine blood glucose levels?

Answer 22

1. Photometers measure a coloured product
2. Biosensors measure electrical current

Question 23

What is the component in biosensors that converts biological responses to signals?

Answer 23

transducer

Question 24

How do biosensor meters work?

Answer 24

1. What blood comes into contact with reagents, redox reaction immediately occurs
2. The electrons generated from the reaction are read as currents by electrodes

Question 25

What makes an ideal biosensor?

Answer 25

1. The biological detection element must be highly specific and stable
2. Reaction should be independent of physical parameters like pH and stirring speed
3. Response should be accurate, precise, reproducible and linear
4. Affordable, small, portable and user friendly
5. Non invasive with fast results

Question 26

What are the classes of biosensors?

Answer 26

1. Catalytic biosensors
2. Affinity biosensors

Question 27

What do catalytic biosensors measure?

Answer 27

They measure change in concentration
1. Rate of product formation
2. Disappearance of a reactant
3. Rate of inhibition of a reaction

Question 28

What do affinity biosensors detect?

Answer 28

A physicochemical change caused by a biomolecule interacting with the targeted analyte

Question 29

What are the types of transducers?

Answer 29

1. Calorimetric – measures heat output (or absorbed) by the reaction.
2. Potentiometric – measures changes in the distribution of charges causing an electrical potential to be produced.
3. Amperometric – movement of electrons produced in a redox reaction (e.g. Glucometer).
4. Optical – light output during the reaction or a light absorbance difference between the reactants and products.
5. Piezo-electric – effects due to the mass of the reactants or products (affinity binding). Generates an electric charge in response to mechanical stress.

Question 30

What is the substance most commonly used in detection elements in biosensors to produce signals that can be detected?

Answer 30

Enzymes

Question 31

How do 1st generation amperometric biosensors work?

Answer 31

1. Glucose is oxidised by oxygen to produce gluconolactone, catalysed by glucose oxidase and cofactor FAD
2. H2O2 is then broken down by the platinum electrode to produce O2, releasing electrons in the process
3. These electrons flow through the electrode and get transduced to form electronic signals

Question 32

How do 2nd generation amperometric biosensors work?

Answer 32

1. Glucose oxidase catalyses the oxidation of glucose and gets reduced in the process
2. The reduced form of glucose oxidase gets oxidised by ferri-cyanide, which gets reduced to ferro-cyanide
3. Ferro-cyanide is oxidised back by the platinum electrode and the lost electrons create a potential which is measured.

Question 33

What is an example of an amperometric biosensor?

Answer 33

Clark oxygen electrode

Question 34

What are the different ways enzymes are immobilised onto the test strip?

Answer 34

1. Adsorption
2. Physical entrapment
3. Covalent bonding

Question 35

How are enzymes immobilised by adsorption into the test strip?

Answer 35

1. Direct absorption into membrane by electrostatic interaction
2. Adsorbed into pre adsorbed proteins like advin and albumin via a biotin linker

Question 36

What is a drawback of using adsorption to immobilise an enzyme to a test strip?

Answer 36

Desorption of proteins

Question 37

How are enzymes immobilised by physical entrapment into the test strip?

Answer 37

1. Lattice type entrapment where the protein is localised within the interstitial space of an
   insoluble matrix
2. Microencapsulation inside liposomes or absorbed in fine carbon particles that are
   incorporated in a gel or membrane.