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1
Q

Why do we perform enzyme assays?

A

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).

2
Q

What considerations that impact enzyme activity?

A

Optimal pH, optimal temp, optimal ionic strength, free from inhibitors, optimal coenzys/cofacs (that influ metab process), and optimal substrate conc (Km).

3
Q

Manometry

A

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.

4
Q

Enthalpimetry

A

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).

5
Q

Spectrofluorimetry

A

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).

6
Q

What is Bioluminescent enzyme assay?

A

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.

7
Q

Electrochemical Methods 1- Potentiometric

A

Electrical potential that is gen is depend upon the conc/property of substance in solution that is undergoing the electrochem rxn.

8
Q

Electrochemical Methods 2- Polarography/Voltammetry

A

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.).

9
Q

Radiochemical Enzyme Assay

A

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.

10
Q

Solid Phase/Dry reagent Enzymatic assays.

A

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).

11
Q

What methods of immobilization for solid phase enzyme assay can be used?

A
  1. Physical adsorption = use of hydrophobic interaction, ionic interaction, van see waals and H bonding.
  2. Covalent binding = using chems to bind the component to the membrane.
  3. I.e. have 96 well plate = have interact btwn the component and the plastic of the well plate which holds it in place.
12
Q

What are the differences between enzyme and chemical drugs?

A
  1. Enzy based drugs are more spec than chem drugs as they have more spec target binding, and so have higher spec.
  2. Enzys target group of molecules and convert it into desired product.
  3. 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.
  4. 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!
13
Q

Name some animal sources of enzymes.

A
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!
14
Q

What are the most common sources for therapeutic enzymes.

A

Microbial sources- Bac

- Fungi

15
Q

Plant sources of enzymes.

A

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.

16
Q

Microbial enzymes that are used in therapeutics and industry.

A

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.)

17
Q

Genetic diseases and therapeutic enzymes.

A

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.

18
Q

Lysozomal Storage Diseases

A
  1. 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).
  2. 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.
19
Q

Sarcosidase- Oral/Inhalable therapy?

A

Oral. Treats congenital sucrase-isomaltase deficiency (CSID). Is a beta-frictohydrolase from Sacchromyces cerevisiae. Enzy enables sucrose hydrolysis allowing norm diet.

20
Q

Phenylase- Oral/Inhalable therapy?

A

Oral. Yeast phenylalanine ammonia phase used. Is as reasonably well-known and researched therapy. Used for treatment of PKU-deficiency of phenylalanine hydroxylase which converts Phenylalanine –> Tyrosine. Here, individuals don’t have the enzy, which is an issue as Phe builds up and becomes toxic–> Phenylketouria = confusion, mental disorders, epilepsy.

21
Q

Pulmozyme (Dornase alpha)- Oral/Inhalable therapy?

A

Inhalable. Routinely used for CF. Need to break down excess thick mucus.

22
Q

Cancer therapeutic enzymes.

A

L-asparginase = Oncolytic enzy
MECHANISM OF ACTION…

  1. Norm tiss synth L-asparginase in sufficient quantities for Protein synth.
  2. Most neoplastic tiss require the exogenous supply from circulation.
  3. L-asparginase breakdowns circulating aa to L-aspartate and ammonia = prevent protein synth of neoplasms = decrease cancer prod by leading to apoptosis.
23
Q

What assaying techniques can be used for enzyme purity?

A
  1. Electrophoresis
  2. Ab mediated ID
  3. Chromatography
24
Q

Assaying for enzyme purity.

A

If the enzy is multimeric (i.e. certain parts of the enzy is suitable for therapies), can ID which subunits of the enzy are working/functional, using MASS SPEC!

An alt method of measuring enzy func is thru the use CIRCULAR DICHROISM SPECTROSCOPY- this method measures the how the diff in conformational changes of the enzy affects its func/activity. Would be able to tell if enzy act gets compromised, as diff peaks are shown on graph.

25
Q

Novel Biocatalyst application.

A
  1. Unique substrate specificities.
  2. Enhanced catalytic acts.
  3. High commercial value.
  4. Effective tools in Bioinformatics.
  5. Green chemistry.
26
Q

Novel Biocatalysts as synthetic tools.

A

Catalysed by enzys from the monoxygenase fam.
These rxns take only one step- something that would otherwise take several steps to reach final product.
Isomers of certain enzys can be differentiatly selected.

27
Q

What do we measure in disease enzymology?

A

Enzys in plasma.

i.e: plasma spec enzys, secreted enzys, cellu enzys.

28
Q

Name some enzyme markers that are looked for in disease.

A
  1. Cell dam
  2. Increased turnover of the cell
  3. Proliferation of the cell
  4. Increased enzy synth
  5. Decreased clearance.
    These markers can sometimes be directly correlated to disease state/progression.
    The half-life of the enzy is depend upon its inact/removal from the body, i.e. AST has a half-life of 17 hrs whereas CPK has a half-life of 15 mins.
29
Q

What do the increased levels in plasma enzymes show?

A
  1. Increased apoptosis/degree of cellu dam
  2. Increased lvs of enzys at intracellu lv for long periods of time
  3. Amount of tiss dam that may have occured
30
Q

How can we ID where the enzyme came from?

A
  1. Can look for enzys that are spec for spec organs (i.e. the liver enzys = Transaminases).
  2. Measure for isoenzys (i.e. lactate dehydrogenase, LDH enzys).
  3. Thru the analysis of several enzy patterns (this will indicate whether there are any issues w/ the organ itself).
31
Q

What are isoenzymes?

A

Isoenzys are enzys that catalyse same rxn however, they, have diff primary structure- which means they have diff phys & chem properties to each other. This allows for the ability to differentiate btwn them when diagnosing disease.

32
Q

Isoenzyme 1: LDH.

A

LDH has 4 subunits that catalyse the same rxn. However, the arrangement of the subunits gives you 5 diff isoenzy variations, all of which have slightly diff properties.

33
Q

LDH isoenzymes methods of measurement.

A

Main method is electrophoresis. The combo of the diff subunits means that when run on SDS PAGE, can see diff in band intensities for each subunit. This will give an indication of what type of LDH isoenzy is present in patient samp and therefore, what organ tiss is dam (i.e. if see higher intensities of LDH-1 & -2 &/-3 = Heart LDH. If see higher band intensities for LDH-3 & -4 = Brain LDH. High intensity of LDH-5 = Muscle LDH. High intensity of LDH-4 & -5 = Retina LDH).

34
Q

Isoenzyme 2: Creatine Kinase.

A

Also called creatine phosphokinase (CPK). Has 3 isoenzys (again formed by combo of diff subunits).
CK1 (BB) = abundant in brain & smooth muscle (practically absent from serum).
CK2 (MB) = abundant in cardiac muscle and some skeletal muscle (again, prac absent from serum).
CK3 (MM) = abundant in skeletal and cardiac muscle (is 100% present in serum).

35
Q

Name 2 CK isoenzyme detection assays.

A
  1. Electrophoretic separation.

2. Column Chromatography.

36
Q

Name some methods for measuring isoenzymes.

A
  1. Immunoassays (spec epitopes of Abs, pick up spec isoforms).
  2. Electrophoresis.
  3. Coenzym analogues (diff isoenzys use slightly diff coenzys for help in cata rxns- can ID what coenzys present, can help ID type of isoenzy present).
  4. Thermostability (diff enzys have diff optimal temps they can w/stand).
  5. Inhibitor sens.
  6. Substrate mods.
37
Q

i.e. what would a graph for serum enzymes show following MI.

A

Tends to be a dramatic increase in the lvs of the enzys present in the serum (espec in LDH, CPK & BHBD) over the first 7 days.

38
Q

i.e. what would a graph for the enzyme profile of Acute Viral Hepatitis (AVH) look like?

A

Hepatitis affects the liver and its func- would therefore expect to see increase in liver enzys (i.e. ALT & AST).
Unlike the graph for MI (which only lasted for max 8 days), this graph shows that the elevation in the enzys lasts for wks (indicating long period of time before get back to more appropriate lvs of enzys act).

39
Q

How would the graph showing the profile of enzymes present in the serum for an individual who has been diagnosed with Acute Alcoholic Hepatitis (AAH) differ from that of AVH?

A

The enzys that are present would be same for both situations (both affecting liver act). However, which enzyms are more act in the situation of AAH will be diff in comparison to AVH, because the dam to the liver tiss will be slightly diff depend upon the situation.

40
Q

What is automation?

A

Automation uses devices that are controlled by humans to control and regulate a process w/out human intervention so that a task can be performed w/ improved efficiency.

41
Q

What are the benefits of using automation?

A
  1. IMPROVED EFFICIENCY- as no need for man labour, can operate constantly, there is reduction in error & increase in precision (which would otherwise be caused by inconsistencies in human techniques), smaller samp/reagent vols (as working in smaller quantities improves precision and therefore reliability of results, it’s also a faster and higher thruput method.
42
Q

What is the main disadvantage of automation?

A

Cost!! - never cheap to buy, setup & maintain.

43
Q

i.e. of manual method that can be automated:

A

Spectrophotometer exp = cuvette w/ samp in which is put in mass spec and the enzy act is measured (thru use of light at spec wavelength).

In automation, there are a range of fluids that will be passed thru & fill cuvettes. Filters are put in place to ensure certain parameters that need to be measured are measured. This process would also take less time than the manual method.

44
Q

What is Continuous Flow Analysis (CFA)?

A

Is a fast, high thruput method that uses kinetics.

45
Q

Principle behind CFA.

A

Inject samp into sys which consists of fast slowing solution in a small-bore tubing. The samp is mixed w/ reagent, which allows for the determination of the samp conc thru change in colour. Carefully controlled flow conditions ensures that colour formation is reproducible.

46
Q

Comparison of CFA to Automated analysers (AA).

A

Unlike CFA, AA keep the samp sep thru/out whole process, only dispensing small precise amounts when nec. Cuvettes are rotated thru the instrument as opposed to letting the samp flow thru in a continuous stream.

47
Q

Name some high throughput enzyme assays.

A
  1. Fluorescence Intensity, including FRET
  2. Fluorescence Polarisation, including FarRed and Transcreener.
  3. Luminescence.
48
Q

Calculating ALT activity in U/L:

A

delta Abs (change in the abs collected)/time (mins) x 6220 (which is the known molar extinction coefficient for NADPH.

49
Q

Calculating Coefficient of Variance (CV%):

A

SD/Mean