Enzyme Assays And Automation Flashcards
Why do we perform enzyme assays?
CLINICAL APPLICATIONS-
Allows us to understand what is going wrong in disease (can mod enzys, I.e. WT mutations, to help understand what goes wrong in disease).
Determinination of disease severity (can allow staging of disease, through using enzys which can act as markers in disease states- enzy assays can give us an understanding of what poss sys have been disrupted/become dysfunc, resulting in disease).
Enzys as drug targets (assoc of enzy w/ disease, target w/ drug = reduce severity of disease).
Enzys as drug molecules (can use enzys as a way of targetted and spec therapy- case if body doesn’t produce enzys/enough of enzys).
USE ENZYMES AS TOOL KITS-
Tools to synth molecules we want and, use them as analytical tools to measure other molecules.
These kits allows for v spec rxns to be catalysed, as can get enzy do whatever we want (decreases labour intensive work in work environ and increases specificity and reliability).
What considerations that impact enzyme activity?
Optimal pH, optimal temp, optimal ionic strength, free from inhibitors, optimal coenzys/cofacs (that influ metab process), and optimal substrate conc (Km).
Manometry
Is a spec technique that is amicable to rxns where one component is in gas state.
Samp and reagents placed in sep compartments, mixed for a known period of time, and then rxn followed as it happens.
Can perform end point and kinetic assays using this method.
I.e. oxygen consumption can be measured in glucose oxidase rxn and carbon dioxide can be measured in rxns which use decarboxylases.
Enthalpimetry
Bases on enthalpy: measure of free energy change in a sys.
V sens technique that is universally applicable method.
Can be adapted to suit variety of methods = SENSITIVE!
No interference would be found as just measuring enthalpy.
Produves reliable measurements:
- has an accurate thermostat sys
- has a highly efficient temp sensor which can detect when temp deviates from optimal.
Can have simple applications = IF thermal imaging.
Can have more complex applications = Hexokinase catalysed rxn (conversion of a hexose sugar –> phosphorylated form using a Hexokinase enzy).
Spectrofluorimetry
Formation of product/the reduction of the reactants conc thru the means of attaching a moiety that fluos at spec wavelength.
Fluo intensity is proportional to intensity of light of approp wavelength.
I.e. Dibytyryl Fluorescein (non-fluo reactant) –> Lipase –> Fluorescein (fluo product).
If- Io x 2.3eclq (key equation!!)
E.g. where can be used: Acute pancreatitis (AP):
Take AP patient blood samp and centrifuge down to get serum samp. Dilute the serum samp and add probe solution to the diluted down serum. What would find is that, individuals who have AP, would have much higher lvs of Lipase enzy in blood than a normal healthy individual. This causes the serum samp to fluo an intense light bluish colour (showing presence of Lipase act).
What is Bioluminescent enzyme assay?
Is an enzy assay that can quantitatively measures the release of an enzy that can be nat found in my human and mammalian cells.
It’s a routinely used method.
Lyse cell thru apoptosis (using complement fixing Abs to form MAC), which releases the enzy. Thru cascade rxn (by where reduce cofac, but oxidase substrate), release ATP (w/ help from externally adding another enzy and enzy-fluorescein complex) = measure light intensity produced- light released is proportional to release of the enzy from cell and is therefore an indication of how much apoptosis occurred.
Electrochemical Methods 1- Potentiometric
Electrical potential that is gen is depend upon the conc/property of substance in solution that is undergoing the electrochem rxn.
Electrochemical Methods 2- Polarography/Voltammetry
Have 2 electrodes, e- flow btwn them.
Increased voltage applied btwn the 2 electrodes that are immersed in test solution. Change in potential measured.
Composition of test solution determines current which flows each time.
The analyte that flows thru, comes into contact w/ biorecep that is impreg on memb. Transducer that is implanted into sys can detect when a sig is produced.
Particular tech is used in toxicology studies (e.g.).
Radiochemical Enzyme Assay
Radioact labelled substrate used to follow the enzy rxn.
Is a highly sens technique, where picomolar concs of reactants and products are measured.
Common isotopes: 3H tritium, 32P phosphorus, 35S sulphur, I131 iodine. ( tritium is not used v much cause of its long 1/2 life).
Principle- enzy rxn performed for defined period of time. It is then quenched using a reagent. Substrate is separated from product in electrophoresis/chromatography. The radioactive fractions of the product/substrate are used to est enzy act.
Solid Phase/Dry reagent Enzymatic assays.
Immobilize one component on solid phase apparatus = develop of portable and easy use, point of care enzy assay kits.
I.e. limitus/pH paper/dipstick papers.
Can be fully quantitative or semi-quantitative estimation of content in fluid/samp.
I.e. detection of D-glucose and Kevin body conc in urine (test for diabetes).
What methods of immobilization for solid phase enzyme assay can be used?
- Physical adsorption = use of hydrophobic interaction, ionic interaction, van see waals and H bonding.
- Covalent binding = using chems to bind the component to the membrane.
- I.e. have 96 well plate = have interact btwn the component and the plastic of the well plate which holds it in place.
What are the differences between enzyme and chemical drugs?
- Enzy based drugs are more spec than chem drugs as they have more spec target binding, and so have higher spec.
- Enzys target group of molecules and convert it into desired product.
- Disadv- they are proteinaceous in nat. This means need to consider administration route since if taken orally, would need to coat so doesn’t get broken down by proteases in stomach.
- Purity and homogeneity is essential after prep of drug = enzys of interior quality that are used for therapies, can cause severe IS responses (I.e. enzy could have multiple binding sites = non-spec binding occur resulting in IS act). Can be resolved by making enzy more spec to func!
Name some animal sources of enzymes.
Lipase- animal pancreas Trypsin- Ox bile Urokinase- human plasma/cow urine Lysozyme- egg whites Adenosine deaminase- Bovine intestine Pepsin- Hog pancreas Dornase alpha- recombinant human cells (useful in immunotherapeutics and pulmonary drugs) = biotech methods!
What are the most common sources for therapeutic enzymes.
Microbial sources- Bac
- Fungi
Plant sources of enzymes.
Plain- Papaya = treat very things such as wounds.
Nattokinase (Nato) = v act enzys that have various therapeutic roles.
Amylase (Malted barley- Hordeum vulgare) = can be easily scaled up on industrial scale.
Bromelain (Ananas Comosus)- used in some cosmetics. Comes from pineapples.
Microbial enzymes that are used in therapeutics and industry.
Beta lactamase = Staphlyococci sp.
Staphylokinase = Staphylococci sp.
Rhodanese = Sulfobacillus sibricus (converts toxic form of cyanide to other intetmeds).
Streptokinase (haemolytic streptococci)
L-asparginase (E.coli)
Collagenase (Clostridium histolyticum)
Amylase (Bacillus sp.)
Genetic diseases and therapeutic enzymes.
I.e. Gaucher (lysozomal storage disease).
Therapeutic enzys that can be used for:
Coagulation diseases = Procoagulants and Anticoagulants.
Cancer = Drug activating enzys, Antineoplastic enzys, Kinase inhibitors.
Infectious diseases = Antibacterial, Antifungal, Antiparasitic.
Lysozomal Storage Diseases
- Fabry’s Disease: Galactosidase A [Gb3]-
Rare genetic disease. Deficiency of enzy alpha-Galactosidase A. Causes the build up of fat calleglobotriaosylceramide (Gb3/GL-3) in body.
Give recombinant human form (GLOBOTRIANOSYLCERAMIDE). - Gauchers disease: Glucocerebrosidase-
Autosomal recessive. Metabolism disorder where type of fat (lipid) called glucocerebroside is not adequately degraded. Builds up on spleen and liver = decrease func.
Sarcosidase- Oral/Inhalable therapy?
Oral. Treats congenital sucrase-isomaltase deficiency (CSID). Is a beta-frictohydrolase from Sacchromyces cerevisiae. Enzy enables sucrose hydrolysis allowing norm diet.
