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Advanced Analytical Techniques > Enzyme Assays and Automation > Flashcards

Enzyme Assays and Automation Flashcards

1
Q

why are enzyme assays performed?

A
  • clinical applications
  • understanding of diseases
  • clinical marker for severity of a disease
  • enzymes as drug targets
  • enzymes as drug molecules
  • enzymes as biotechnology tool kits
    • tools to synthesise molecules
    • analytical tools to measure other molecules
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2
Q

what needs to be taken into consideration when following an enzyme reaction?

A
  • optimal pH
  • optimal ionic strength
  • optimal temperature
  • free from inhibitors
  • optimal coenzymes/cofactor concentrations
  • optimal concentration of the enzyme (Km)
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3
Q

detection techniques used in enzyme activity assays:

A

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

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4
Q

What is spectrophotometry

A

Spectrophotometry is the quantitative measurement of the interaction of ultraviolet (UV), visible, and infrared (IR) radiation with a material

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5
Q

What is calorimetry?

A

measures colour change, e.g., albumins, carbs, produce colour. Intensity of colour proportional to concentration

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6
Q

What is used in spectrofluorimetry?

A

fluorophore is used

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7
Q

the formation of product or reduction of reactant concentration is measured…

A

by attaching a moiety that fluoresces at a defined wave length

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8
Q

What happens at low concentrations?

A

the fluorescent intensity is related to the intensity of light,

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9
Q

Examples of spectrofluorimetry

A
  • dibutyryl fluorescein
  • intrinsic fluorescent amino groups - tyrosine
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10
Q

What condition can it be used for?

A
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11
Q

What equation is used to calculate spectrofluorimetry?

A
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12
Q

antibody dependent cell-mediated cytotoxicity (ADCC) what does this measure?

A

quantitatively measures release of glyceraldehyde-3-phosphate dehydrogenase (GADPH) found in mammalian cells
- The lysis of the cell causes the release of the enzyme, allowing the detection of cell destruction within patients

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13
Q

What type of technique is manometry?

A

Monometric technique

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14
Q

What is manometry used to measure?

A

measures enzyme activity if one of the components is in gaseous form.

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15
Q

Two different compartments in the device, what do they do?

A
  • one contains the gas from the reaction
  • the other is where the reaction takes place
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16
Q

What happens to the samples and reagents?

A

samples and reagents are placed in separate compartments, they are then mixed at defined time periods and the reaction is followed as the reaction proceeds
both end-point and kinetic assays can be performed

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17
Q

Examples of manometry

A
  • glucose oxidase enzyme:
    • oxygen consumption is measured
  • decarboxylase enzymes:
    • carbon dioxide production is measured
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18
Q

electrochemical methods (what are the two methods?)

A

Potentiometric techniques
Polarography/voltammetry

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19
Q

What are potentiometric techniques

A

there is an electrical potential generated that is dependent on the concentration/properties of the substances in the solution that is undergoing an electrochemical reaction

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20
Q

What does polarography/voltammetry do?

A

increased voltage is applied between two electrodes immersed in a test solution and the change in potential is measured

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20
Q

The composition of the test solution determines what?

A

the current which flows at each instance

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21
Q

What does enthalpimetry measure?

A

measure the enthalpy change during the reaction - heat change is measured by sensitive and efficient temperature sensor

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22
Q

what is needed in this system to maintain accuracy?

A

excellent insulation

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23
Q

what are the advantages of this technique?

A
  • sensitive
  • easily adaptable for various applications
  • freedom from interference
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24
Q

give an example of a reaction that can be measured with enthalpimetry?

A

Hexokinase catalysed reaction

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25
Q

Radiochemical methods

A
  • radioactively labelled substrate is used to follow the enzymatic reaction
  • it is very sensitive - can be measured to picomolar concentration
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26
Q

Protocol

A

the enzyme reaction is performed for a defined period and quenched using a reagent this is to stop the reaction
the substrate is then separated from the product using electrophoresis or chromatography

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27
Q

What can the radioactive fraction of the product/substrate be used to estimate?

A

the activity of the enzyme

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28
Q

common isotypes used:

A

3 H - tritium

32 P - phosphorus

35 S - sulphur

131 I - Iodine

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29
Q

solid phase/dry reagent enzymatic assay

A
  • there is the immobilisation of components onto a solid phase, resulting in the production of portable, easy to use and point of care enzymatic assay kits
  • they can be fully/semi-quantitative
  • there can be the assay of various enzyme activity on plates with solid medium that has the corresponding substrates that is needed for the detection of various enzymes
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30
Q

what are the different methods for immobilisation for a solid phase enzyme assay?

A
  • physical adsorption:
    • hydrophobic interaction
    • ionic interaction
    • van der waals’ force
    • hydrogen bonding
  • covalent binding:
    • DSS
    • EDC
    • NHS
    • Maleimide
    • hydrazine
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31
Q

what are the differences between enzyme and chemical drugs?

A
  • enzymes are more natural - biological molecules
  • enzymes are highly specificwill only act on target molecules unlike chemicals
  • enzymes are more predictablewe know which site they will bind to
  • enzymes convert targeted substances into desired products
  • as enzymes are proteins they will need enteric coating for oral administrationthis will prevent them from being broken down in the body
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32
Q

the enzymes need to be very pure, why?

A

they can cause allergic reactions in patients

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33
Q

what are the different sources for therapeutic enzymes?

A
  • animal sources
  • plant sources
  • microbial sources
    • bacterial
    • fungi
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34
Q

common animal source enzymes: where are they sourced from?

A
  • lipaseAnimal pancreas
  • trypsinOx bile
  • urokinasehuman plasma/cow urine
  • lysozymemilk/chicken albumen
  • adenosine deaminasebovine intestine
  • pepsinhog pancreas
  • dornase alpharecombinant human cells
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35
Q

plant sourced enzymes:

A
  • papainpapaya
  • nattokinasenatto bean
  • amylasebarley
  • bromelainpineapple
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36
Q

bacterial 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.
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37
Q

bacterial 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.
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38
Q

what is the preferred source of enzymes? Why?

A

Microbes because :
- cheaper to produce
- content of enzymes can be:
- estimated
- controlled
- reliable supply of raw material of constant composition
- other sources contain more harmful:
- phenolic compounds
- endogenous inhibitors
- proteases

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39
Q

novel bio catalyst application:

A
  • unique substrate specificities - new enzymes made?
  • enhanced catalytic activities - more efficient
  • high commercial value
  • effective tools in biotransformation
  • green chemistry
  • biocatalyst as a synthetic tool:
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40
Q

name two lysosomal storage diseases:

A

Fabry’s disease
Gauchers disease

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41
Q

fabry’s disease

A
  • galactosidase A [Gb3], used to break down what?used to break down a particular type of fat
  • Deficiency in whatthis is a disease due to a deficiency in the enzyme alpha-galactosidase A
  • Causes a buildup in a fat calledglobotriaosylceramide in the body - this is a complex lipid
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42
Q

Gauchers disease:

A
  • glucocerebrosidase
    • used to break down a particular type of fat
  • autosomal recessive inherited disorder of metabolism where a lipid called glucocerebroside cant be degraded
    Build up in the liver and spleen
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43
Q

Oral and inhalable therapies:

A
  • sarcosidase (oral):
    • the enzyme enables sucrose hydrolysis, allowing for a normal diet
    • taken orally to treat congenital sucrase-isomaltase deficiency (CSID)
    • it is a b-fructofuranoside fructohydrolase from sacchromyces cerevisiae
  • Phenylase (oral) :
    • Yeast Phenylalanine ammonia-lyase is used
    • used for the treatment of PKU-deficiency of phenylalanine hydroxylase
    • what does this convert?
      phenylalanine to tyrosine
  • Pulmozyme (inhalable):
    • used in cystic fibrosis treatment management
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44
Q

cancer therapeutic enzymes:

A

L-asparaginase - oncolytic enzyme

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45
Q

Mechanism of action of L-asparaginase - oncolytic enzyme

A
  • normal tissues synthesise L-Asparagine in sufficient quantities for protein synthesis
  • neoplastic tissues require exogenous supply from circulation
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46
Q

L-aparaginase breaksdown circulating aa (L-asparagine) to what?

A

L-aspartate and ammonia which prevents protein synthesis of neoplasm → apoptosis

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47
Q

other important therapeutic enzymes:

A
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48
Q

What is the function of Lipase?

A

It breaks down fat.

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49
Q

Where can you find Lipase?

A

It is found in the pancreas, mouth and stomach.

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50
Q

How is Lipase usually sourced.

A

It is usually sourced from animal pancreas.

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51
Q

What is the function of Trypsin?

A

Helps to digest proteins by cleaving them.

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52
Q

Where can Trypsin be found?

A

It is found in the small intestine.

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53
Q

How is Trypsin sourced?

A

Ox bile

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54
Q

What is the function of Urokinase?

A

Converts inactive plasminogen into active plasmin.

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55
Q

Where is Urokinase located?

A

It is made in the kidney and found in urine

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56
Q

How is Urokinase sourced?

A

Human Plasma/Cow Urine

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57
Q

What is the function of Lysozyme?

A

It is an antimicrobial agent by cleaving the peptidoglycan components of bacterial cell walls which leads to cell death

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58
Q

Where is Lysozyme located?

A

Found in bodily secretions such as tears, saliva and milk

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59
Q

How is Lysozyme sourced?

A

Transgenic plants, animals and microorganisms that can produce human lysozyme

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60
Q

What is the function of Adenosine Deaminase?

A

It eliminated deoxyadenosine which is generated when DNA is broken down which is toxic to lymphocytes to deoxyinosine which is not harmful.

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61
Q

Where is Adenosine Deaminase located?

A

Produced in all cells however mostly in lymphocytes

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62
Q

How is Adenosine Deaminase sourced?

A

Bovine Intestine

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63
Q

What is the function of Pepsin?

A

Breaks down proteins in food during digestion

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64
Q

Where is Pepsin located?

A

Stomach

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65
Q

How is Pepsin sourced?

A

Hog pancreas

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66
Q

What is the function of Dornase a?

A

Is an inhaled medication that thins mucus. Reduces the number of lung infection and to improve lung function in patients with cystic fibrosis

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67
Q

Where is Dornase a located?

A

Medication

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68
Q

How is Dornase a sourced?

A

It is a highly purified solution of recombinant deoxyribonuclease I (rhDNase) an enzyme which actively cleaves DNA.

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69
Q

What is the function of Beta Lactamase?

A

Stop bacterial growth by inhibiting PBPs that are indispensable for the cross linking process during cell wall biosynthesis. This may also lead to antibiotic resistance as mutations of B-lactamase active site, leading to ineffective binding and thus reduced inhibition.

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70
Q

What is the function of Staphylokinase?

A

Activates plasminogen to form plasmin which digest fibrin clots.

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71
Q

What is the function of Rhodanese?

A

Main enzyme in sulfur metabolism for cyanide detoxification & anti-oxidative stress systems.

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72
Q

What is the function of Streptokinase?

A

Dissolve blood clots that have formed in the blood vessels. Used to treat blood clots in the lungs (pulmonary embolism) and in the legs (deep venous thrombosis)

73
Q

What is the function of L-aspariginase?

A

Catalyses the hydrolysis of the non-essential amino acid L-asparagine to L-aspartate and ammonia. Used for the treatment of haemopoiertic diseases such as ALL( acute lymphoblastic leukaemia).

74
Q

What is the function of collagenase?

A

Break the peptide bonds in collagen. They assist in destroying extracellular structures in the pathogenesis of bacteria such as Clostridium.

75
Q

What is the function of amylase?

A

Helps you digest carbohydrates. It is used when the pancreas cannot make or does not release enough digestive enzymes into the gut to digest the food.

76
Q

What is the Principle behind Asparaginase?

A

Reduces the availability of asparagine, an amino acid that is necessary for the growth of some tumour cells.

77
Q

What reaction does Asparaginase do?

A

Asparagine conversion

78
Q

What are the therapeutic uses of Asparaginase?

79
Q

What is the Principle behind Collagenase?

A

Cleaves collagen

80
Q

What reaction does Collagenase do?

A

Collagen hydrolysis

81
Q

What are the therapeutic uses of Collagenase?

A

Skin ulcers

82
Q

What is the principle behind Hyaluronidase?

A

Considered a ‘spreading factor; as it decomplexes hyaluronic acid, an essential component of the extracellular matrix. When applied as an adjuvant, hyaluronidase enhances the diffusion capacity and bioavailability of injected drugs.

83
Q

What reaction is Hyaluronidase responsible for?

A

Hyaluronate hydrolysis

84
Q

What are the therapeutic uses of Hyaluronidase

A

Heart Attack

85
Q

What is the principle behind Ribonuclease?

A

Cleaves RNA

85
Q

What is the principle behind Ribonuclease?

A

Cleaves RNA

86
Q

What reaction is Ribonuclease responsible for?

A

RNA Hydrolysis

87
Q

What is the therapeutic use of Ribonuclease?

A

Anti Viral

88
Q

What is the principle behind Streptokinase?

A

Creates an active complex which promotes the cleavage of the Arg/Val bond in plasminogen to form the proteolytic enzyme plasmin.Plasmin in turn degrades the fibrin matrix of the thrombus, thereby exerting its thrombolytic action.

89
Q

What reaction is Streptokinase responsible for?

A

Plasminogen to Plasmin

90
Q

What is the therapeutic use of Streptokinase?

A

Blood Clot

91
Q

What is the principle behind Uricase?

A

Pegloticase, a recombinant mammalian uricase modified with monomethoxylpoly (ethylene glycol)(mPEG) is effective in treating refractory gout

92
Q

What reaction is Uricase responsible for?

A

Urate to Allantoin

93
Q

What is the therapeutic use of Uricase?

94
Q

What is the principle behind Urokinase?

A

Urokinase-type plasminogen activator, is a serine protease present in humans and other animals.

95
Q

What reaction is Urokinase responsible for?

A

Plasminogen to Plasmin

96
Q

What is the therapeutic use of Urokinase?

A

Blood Clot

97
Q

What is the principle behind Ribonuclease L?

A

RNase L is an enzyme found in all cells that is activated when a cell is under attach by viruses, some bacteria and at least some toxins

98
Q

What reaction is Ribonuclease L responsible for?

A

RNA Hydrolysis

99
Q

What is the therapeutic use of Ribonuclease L?

A

Chronic Fatigue Syndrome

100
Q

assaying for enzyme purity ( 2 tests)

A
  • test for homogeneity:
    • electrophoresis
    • antibody mediated identification
    • chromatography
    • mass spectrometry
      • can test for presence of required subunits
  • testing efficiency:
    • the enzymes function can be determined
101
Q

what enzymes are measured in the plasma?

A
  • plasma specific enzymes
  • secreted enzymes
  • cellular enzymes
102
Q

when can enzymes be raised in the plasma?

A
  • increased cell damage
  • increased cell turnover
  • proliferation of cells
  • increased enzyme synthesis
  • decreased clearance
103
Q

what is the half life of an enzyme dependent on?

A
  • the enzymes inactivation
  • the enzymes removal
104
Q

what determines the levels of enzymes in the plasma?

A
  • degree of damage
  • original intracellular levels
  • amount of tissue that is affected
105
Q

how can the enzymes original location be identified?

A
  • measure organ specific enzymes
  • measure isoenzymes
  • analysis of patterns of several enzymes
106
Q

organ specific enzyme examples:

A
  • liver enzymes - transaminases (AST + ALT)
  • pancreatic enzymes - pancreatic lipase + amylase
107
Q

what are isoenzymes?

A
  • catalyse the same reaction
  • different primary structure
  • different physical/ chemical properties
  • differentiated
108
Q

isoenzyme examples

A

Lactate Dehydrogenase
Creatine Kinase

109
Q

What is the method used to measure lactate dehydrogenase?

A

Electrophoresis it gives you 5 different isoforms

110
Q

Was is Creatine Kinase the same as?

A

creatine phosphokinase (CPK)

111
Q

Creatine Kinase has 3 isoenzymes formed by combinations of different subunits what are they? Where are they abundant?

A
  • CK1 (BB)
    • abundant in brain and smooth muscle (practically absent from serum)
  • CK2 (MB)
    • abundant in cardiac muscle, some in skeletal muscle (practically absent from serum)
  • CK3 (MM)
    • abundant in skeletal muscle and cardiac muscle (practically 100% of serum CK)
112
Q

How do you detect CK iso-enzyme?

A
  • electrophoretic separation
  • column chromatography
113
Q

How do you measure an isoenzyme? 6 different techniques

A
  • electrophoresis
  • inhibitor sensitives
  • modified substrates
  • coenzyme analogues
  • thermostability
  • immunoassay - this is if there is only slight differences between the isotypes
114
Q

serum enzymes following myocardial infarction how does the graph look

115
Q

enzyme profile for acute viral hepatitis graph explain

116
Q

enzyme profile for acute alcoholic hepatitis explain graph

117
Q

what is automation?

A

when a process is carried out by a device that is controlled by humans to perform it with a higher degree of efficiency

118
Q

what is a semi-automatic device?

A

this is a device that is controlled by humans with a higher degree of human involvement than an automatic device

119
Q

what are the benefits of automation? 7 benefits

A
  • no need for manual labour
  • operate continuously
  • reduced errors
  • improved precision and accuracy
  • smaller sample/reagent volumes can be used
  • faster
  • can sometimes be cheaper - does depend on the machines price and maintenance
120
Q

how are automatic machines developed?

A
  1. need for a method
  2. method designed
  3. increased throughput needed for the method
  4. automated system developed
121
Q

first instrument for enzyme analysis:

122
Q

What is LKB?

A

an automated machine that has two different compartments
- one containing the substrate
- other containing buffers and substrates that are required for the reaction

123
Q

samples are pipetted using what in a LKB machine?

A

a semi-automatic pipettor into a cuvette - on the bottom of the cuvette is a star shape groove

124
Q

What happened to the cuvette containing the sample in a LKB machine?

A

the cuvette containing the sample is loaded into a rack, the rack is then loaded into the machine - when it is started a mechanical feed moves the rack through the instrument

125
Q

What will happen when the sample reaches the nozzle?

A

it will then reach the nozzle for buffer and the correct amount will be added using automated syringes

126
Q

Why is the substrate added?

A

the substrate is then added to start the reaction and the absorbance is then measured instantly - there is effectively a spectrometer within the machine.

127
Q

Benefits of LKB

A
  • there will be the same treatment for every sample
  • reduced errors
    • increased accuracy
    • increased precision
128
Q

Drawbacks of LKB

A
  • the machine was relatively slow - 12 tests per hour
  • racks were forever sticking - buffer/ substrate would be repeatedly added to the same cuvette
  • when the cuvettes were full, the spinning stage would cause the inside of the machine to be covered in potentially infectious sample
129
Q

What has automation done?

A
  • replaced all the manual steps - reduced manual labour
  • walk-away capability - a technician isn’t needed at all times
  • increased reliability
130
Q

What is a continuous flow analysis?

A
  • rapid determination of a well defined enzyme reaction
  • kinetics had to be well defined
  • there are a number of large scale/high throughput analyses in action now
  • is of little use for true kinetic studies
131
Q

What is the concept behind continuous flow analysis?

A
  • instead of the samples being added to test tubes, they are added to just one tube, which the sample and reagents are all run though
    • there is a continuous flow through the tube which is then passed through a mixing coil
  • if the reaction needs to be incubated then the tube will be passed through a water bath set to the required temperature
  • at the end of the reaction the tube will flow into a cuvette which will be analysed by a spectrophotometer within the device which will take a reading of its absorbance - this will always be at the same time, as long as the length of the tube remains constant
  • the absorbance can be measured at different points throughout the machine to give different readings at different time points
  • the information gained can be plotted manually or by the computer
132
Q

How does the coil work in a continuous flow analysis?

A

gravity does the mixing within the coil

133
Q

there are T-junctions added throughout the tube at locations where…

A

reagent might need to be added

134
Q

The devices are limited by processing power. What is the rule?

A

the more processing power there is increases the throughput of the device

135
Q

Why are automated analysers good? 5 points

A
  • ideal for kinetic studies
  • must be computer controlled
  • discrete analysis
  • disposable
  • fast
136
Q

What are the biological applications of automation?

A
  • haemolysis - turbidity
  • lipaemia
  • the sample that is added to the machine also needs to be of good quality as well or results will be inaccurate
137
Q

What are the lag phase considerations of automation?

A
  • it takes time for the reaction to get going
  • is pointless making readings at the earlier stages of the reaction
  • delays can be programmed in before readings are started
138
Q

What is the automatic blanking consideration?

A
  • the machine uses a blank and subtracts it from the rest measurements - this increases the accuracy of the machine
  • this give the true rate of reaction of the enzyme
139
Q

What are the common techniques for immobilisation of enzymes?

A

Absorption
Entrapment
Covalent Bonding
Cross Linking

140
Q

What is adsorption?

A
  • Physical binding of enzymes (or cells) on surface of an inert support
  • Support may be inorganic or organic
141
Q

What happens on inert support?

A

Involves weak forces such as Van der Waals forces and hydrogen bonds

142
Q

How can the adsorbed enzymes be easily removed?

A

By minor changes in pH, ionic strength or temperature (this is a disadvantage for industrial use of enzymes)

143
Q

Enzymes can be immobilised by physical entrapment inside what?How does this work?

A

A polymer or a gel matrix
Size of the matrix pores is such that the enzyme is retained whilst the substrate and product molecules pass through

144
Q

Enzyme or cell not subjected to what?

A

Strong binding forces and structural distortions

145
Q

How would some deactivation occur during immobilisation?

A

due to changes in pH/temperature/addition of solvents

146
Q

What matrices are used for entrapping enzymes?

A

Polyacrylamide gel
collagen
gelatine
starch
cellulose
silicone
rubber

147
Q

Where are the covalent bonds found?

A

Between the chemical groups of enzymes and the chemical groups of support

148
Q

Disadvantage of covalent bonding

A

Loss of enzyme activity

149
Q

Inert support requires what?

A

pre-treatment (to form pre-activated support) before it binds to enzyme

150
Q

Cross-linking has an absence of what?

A

solid support

151
Q

Enzyme molecules are immobilised how?

A

By creating cross links between them, through involvement of poly functional reagents

152
Q

What do poly functional reagents do?

A

react with the enzyme molecules and create bridges which form the backbone to hold enzyme molecules

153
Q

What is the most common poly functional reagent?

A

Glutaraldehyde

154
Q

How does Glutaraldehyde reagent work?

A

React with lysyl residues of the enzymes and forms a Schiff’s base

155
Q

Cross links formed between the enzyme and glutaraldehyde are…

A

irreversible and can withstand extreme pH and temperature

156
Q

The cross links formed between the enzyme and glutaraldehyde are used to what?

A

immobilise several industrial enzymes

157
Q

Advantages of glutaraldehyde + enzyme linkage

A

Simple
Cost effective

158
Q

Disadvantages of glutaraldehyde + enzyme linkage

A

involves the risk of denaturation of the enzyme by the poly functional reagent

159
Q

Lipase

A

Family of enzymes that break down triglycerides into free fatty acids and glycerol

160
Q

Where is Lipase found?

A

In animal pancreas

161
Q

What does Trypsin do?

A

Helps us digest protein

162
Q

Where can Trypsin be found?

A

in the small intestine

(And Ox bile)

163
Q

Trypsin breaks down what?

A

proteins, continuing the process of digestion that began in the stomach

164
Q

Trypsin is a type of what enzyme?

A

proteolytic enzyme or proteinase

165
Q

where is Trypsin produced?

A

in the pancreas

166
Q

what is inactive Trypsin form called?

A

trypsinogen - produced in the pancreas

167
Q

how does Trypsinogen enter the small intestine?

A

through the common bile duct and is converted into active trypsin

168
Q

what does the active trypsin do?

A

acts with the other two digestive proteinases - pepsin and chymotrypsin

169
Q

what happens after trypsin acts with the other two digestive proteinases - pepsin and chymotrypsin?

A

break down dietary protein into peptides and amino acids

170
Q

what are amino acids essential for?

A

muscle growth, hormone production and other important bodily functions

171
Q

Complications of inadequate trypsin levels

A

Malabsorption

Pancreatitis

172
Q

Alanine transaminase (ALT) what reaction does it catalyse?

A

alanine + a-ketoglutarate → pyruvate + glutamate

173
Q

what secondary reaction is used to determine the activity of the ALT enzyme?

A

lactate dehydrogenase reaction
pyruvate + NADH + H → lactate + NAD

174
Q

what substrate has an absorbance that can be measured?

A

NADH - its consumption is equal to the production of pyruvate which is limited by the activity of the ALT enzyme

@340nm

175
Q

how is the activity of an enzyme determined from the change in absorbance?

A
  • it is determined using the molar extinction coefficient

enzyme activity (U/L) = change in absorbance/min X molar extinction coefficient

176
Q

how is the coefficient of variance calculated?

A

CV= mean/SD

177
Q

how is percentage coefficient of variance calculated?

A

CV (%)= mean/SD X 100

178
Q

what does a low variance mean?

A

the data collected has high precision

Advanced Analytical Techniques (8 decks)

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