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AP0609 - Advanced Analytical Techniques > Enzyme Assays and Automation > Flashcards

Enzyme Assays and Automation Flashcards

1
Q

What do enzyme assays measure?

A
  • either the consumption of substrate or production of product over time
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2
Q

Considerations for an enzyme reaction

A
  • optimal pH
  • optimal ionic strength
  • optimal temperature
  • free from inhibitors
  • optimal coenzymes/ cofactors
  • optimal substrate concentration (Km)
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3
Q

Detection Techniques used in Enzyme activity assays

A

-Spectrophotometry
- Calorimetry
- Spectrofluorimetry
- Manometry
- Electrochemical Methods
- Enthalpimetry
- Radiochemical methods
- Dry-reagent techniques

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4
Q

What is Manometry?

A
  • useful to measure enzyme activity if one of the component is in gaseous form i.e. Oxidases (O2 uptake), Decarboxylase (CO2 output)
  • samples and reagents are placed in separate compartments and mixed at defined time period and the reaction can be followed as the reaction proceeds
  • Both end point and kinetic assays can be performed
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5
Q

What is Enthalpimetry?

A
  • Measures the enthalpy (heat energy) change during the course of a reaction
  • Extremely accurate thermostatting or excellent insulation is required
  • Highly efficient temperature sensor is a must
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6
Q

What makes Enthalpimetry a useful technique?

A
  • added advantage of sensitivity, easily adapted for various applications, freedom from interference
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7
Q

What is Spectrofluorimetry?

A
  • formation of product or reduction of reactant concentration is measured by attaching a moiety that fluoresce at defined wave lengths
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8
Q

Fluorescent enzyme assay to assess Acute Pancreatitis

A
  • in both AP and healthy people; the blood is extracted, centrifuged to make a serum sample, diluted serum sample added to probe solution, probe solution is incubated
  • AP people; strong emission of fluorescence
  • healthy people; weak or no emission
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9
Q

Bioluminescent enzyme Assay to measure antibody dependent cell-mediated cytotoxicity (ADCC)

A
  • Assay quantitatively measures release of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) naturally found in primary, mammalian and bacterial cells
  • In this manner, pre-labeling, transfection, transformation and other methods to artificially introduce molecules into cells is negated. The reagents may be used for CDC and ADCC assays
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10
Q

What Electrochemical methods are used for enzyme assays?

A
  • Potentiometric Techniques
  • Polarography/Voltammetry
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11
Q

What are Potentiometric Techniques?

A
  • Electrical potential generated is dependent on the concentration or property of the substance in solution that is undergoing an electrochemical reaction
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12
Q

What is Polarography/Voltammetry?

A
  • Increased Voltage is applied between two electrodes immersed in a test solution and the change in potential is measured.
  • The composition of the test solution determines the current which flows at each instance
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13
Q

What is Radiochemical Enzyme assay?

A
  • Radioactively labelled substrate is used to follow the enzymatic reaction
  • Highly sensitive where picomolar concentration of reactants and products can be measured
  • Common radioisotopes used for labelling include 3H (tritium), 32P(Phosphorus), 35 S (Sulphur) and 131I (Iodine)
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14
Q

How does Radiochemical Enzyme assay?

A
  • enzymatic reaction is performed for a defined period and quenched using a reagent
  • substrate is then separated from the product using electrophoresis or chromatography and the radioactive fraction of the product or the substrate is used to estimate activity of the enzyme
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15
Q

What are Solid Phase/ Dry reagent Enzymatic Assays?

A
  • Immobilising the components on a solid phase results in developing-portable, easy to use and point of care enzymatic assay kits
  • Fully quantitative or semi-quantitative estimation of the content in a liquid or sample can be measured
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16
Q

Common animal source enzymes

A
  • Lipase - Animal Pancreas
  • Trypsin - Ox Bile
  • Urokinase - Human Plasma / Cow Urine
  • Lysozyme - Eggs
  • Adenosine Deaminase – Bovine intestine
  • Trypsin – Hog Pancreas
  • Dornase α (recombinant human cells)
17
Q

Plant source enzymes

A
  • Papain (Carica Papaya)
  • Nattokinase (Natto)
  • Amylase (Malted barley-Hordeum vulgare)
  • Bromelain (Ananas Comosus)
18
Q

Bacterial source enzymes

A
  • Beta lactamase (Staphylococci sp.)
  • Staphylokinase (staphylococci sp.)
  • Rhodanese (Sulfobacillus sibiricus)
  • Streptokinase (hemolytic streptococci)
  • L-aspariginase (E.coli)
  • Collagenase (Clostridium histolyticum)
  • Amylase (Bacillus sp.)
19
Q

What make microbial enzymes a preferred source?

A
  • Cheaper to produce
  • Content of enzyme –estimated, controlled
  • Reliable supply for raw material of constant composition
  • Other sources contain more harmful phenolic compounds,endogenous Inhibitors and proteases
20
Q

Applications of novel biocatalysts

A
  • Unique Substrate specificities
  • Enhanced catalytic activities
  • High commercial Value
  • Effective tools in Biotransformation
  • Green Chemistry
21
Q

What is Fabry’s disease and what enzyme is used as a therapeutic?

A
  • galactosidase A
  • rare genetic disease a deficiency of the enzyme alpha-galactosidase A (a-Gal A) that causes a build up of a type of fat called globotriaosylceramide (Gb3, or GL-3) in the body
22
Q

What is Gaucher’s disease and what enzyme is used as a therapeutic?

A
  • Glucocerebrosidase
  • autosomal recessive inherited disorder of metabolism where a type of fat (lipid) called glucocerebroside cannot be adequately degraded, so it builds up in bone marrow, spleen and liver
23
Q

Sarcosidase as an oral therapy

A
  • Taken orally to treat congenital sucrase-isomaltase deficiency(CSID)
  • It is a β-fructofuronoside fructohydrolase from Sacchromyces cerevisiae
  • enzyme enables sucrose hydrolysis allowing normal diet
24
Q

Phenylase as an oral therapy

A
  • Yeast Phenylalanine ammonia lyase is used
  • Used for the treatment of PKU- deficiency of phenylanine hydroxylase which converts Phenylalanine to Tyrosine
25
Q

What is Pulmozyme?

A
  • aka Dornase alpha
  • inhalable therapy
  • used in cystic fibrosis management
26
Q

L-Asparaginase as a cancer therapeutic enzyme

A
  • Normal tissues synthesise L-Asparagine in sufficient quantities for Protein synthesis
  • Most neoplastic tissues require the exogenous supply from circulation
  • L-Asparaginase breaks down circulation aa to L-aspartate and ammonia- prevents protein synthesis of neoplasms (apoptosis)
27
Q

When are levels of enzymes in plasma raised?

A
  • cell damage
  • increased cell turnover
  • proliferation of cells
  • increased enzyme synthesis
  • decreased clearance
28
Q

How to determine level of increase in enzymes?

A
  • degree of damage
  • original intracellular levels
  • amount of tissue affected
29
Q

How to identify where the enzyme came from

A
  • Measure organ specific enzyme (e.g liver has transaminases, pancreas has amylase and lipase)
  • Measure isoenzyme
  • Analysis of patterns of several enzymes
30
Q

What are isoenzymes?

A
  • catalyse the same reaction
  • different primary structure
  • different physical / chemical properties
  • differentiated
31
Q

Lactate dehydrogenase (LDH) as an isoenzyme

A
  • enzyme that the body uses during the process of turning sugar into energy for your cells to use
  • found in muscles, liver, heart, pancreas, kidneys, brain and blood cells
  • measured using electrophoresis (each has different electrophoretic mobility; LDH1 is fastest moving fraction towards anode, LDH5 is slowest)
32
Q

Creatine kinase (CK) or Creatine phosphokinase (CPK)’s 3 isoenzymes

A
  • CK1 (BB) is abundant in brain and smooth muscle (practically absent form serum)
  • CK2 (MB) is abundant in cardiac muscle, some inskeletal muscle (practically absent from serum)
  • CK3 (MM) is abundant in skeletal muscle and cardiac muscle (practically 100% of serum CK)
33
Q

Methods of measurement of isoenzymes

A
  • Electrophoresis
  • Inhibitor sensitivities
  • Modified substrates
  • coenzyme analogues
  • Thermostability
  • Immunoassay
34
Q

What is the definition of an automated device?

A
  • device that is able to control and regulate a process without human intervention, using integrated smaller mechanical/electronic devices, sensors and some kind of feedback loop
35
Q

Benefits of automation

A
  • Improved efficiency
  • No need for manual labour
  • Operate continuously
  • Reduce errors
  • Improved precision
  • Smaller sample/reagent volumes
  • Faster, Cheaper

AP0609 - Advanced Analytical Techniques (10 decks)

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