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
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)
3
Q
detection techniques used in enzyme activity assays:
A
Spectrophotometry
Calorimetry - colour reaction
Spectrofluorimetry
Manometry
Electrochemical methods
Enthalpimetry
Radiochemical methods
Dry-reagent techniques
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
5
Q
What is calorimetry?
A
measures colour change, e.g., albumins, carbs, produce colour. Intensity of colour proportional to concentration
6
Q
What is used in spectrofluorimetry?
A
fluorophore is used
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
8
Q
What happens at low concentrations?
A
the fluorescent intensity is related to the intensity of light,
9
Q
Examples of spectrofluorimetry
A
- dibutyryl fluorescein
- intrinsic fluorescent amino groups - tyrosine
10
Q
What condition can it be used for?
A
11
Q
What equation is used to calculate spectrofluorimetry?
A
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
13
Q
What type of technique is manometry?
A
Monometric technique
14
Q
What is manometry used to measure?
A
measures enzyme activity if one of the components is in gaseous form.
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
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
17
Q
Examples of manometry
A
- glucose oxidase enzyme:
- oxygen consumption is measured
- decarboxylase enzymes:
- carbon dioxide production is measured
18
Q
electrochemical methods (what are the two methods?)
A
Potentiometric techniques
Polarography/voltammetry
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
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
20
Q
The composition of the test solution determines what?
A
the current which flows at each instance
21
Q
What does enthalpimetry measure?
A
measure the enthalpy change during the reaction - heat change is measured by sensitive and efficient temperature sensor
22
Q
what is needed in this system to maintain accuracy?
A
excellent insulation
23
Q
what are the advantages of this technique?
A
- sensitive
- easily adaptable for various applications
- freedom from interference
24
give an example of a reaction that can be measured with enthalpimetry?
Hexokinase catalysed reaction
25
Radiochemical methods
- radioactively labelled substrate is used to follow the enzymatic reaction
- it is very sensitive - can be measured to picomolar concentration
26
Protocol
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
27
What can the radioactive fraction of the product/substrate be used to estimate?
the activity of the enzyme
28
common isotypes used:
3 H - **tritium**
32 P - **phosphorus**
35 S - **sulphur**
131 I - **Iodine**
29
solid phase/dry reagent enzymatic assay
- 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
30
what are the different methods for immobilisation for a solid phase enzyme assay?
- physical adsorption:
- hydrophobic interaction
- ionic interaction
- van der waals' force
- hydrogen bonding
- covalent binding:
- DSS
- EDC
- NHS
- Maleimide
- hydrazine
31
what are the differences between enzyme and chemical drugs?
- **enzymes** are more **natural** - biological molecules
- **enzymes** are **highly specific**
will only act on **target molecules** unlike chemicals
- **enzymes** are **more predictable**
we 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 administration**
this will prevent them from being broken down in the body
32
the enzymes need to be very pure, why?
they can cause allergic reactions in patients
33
what are the different sources for therapeutic enzymes?
- animal sources
- plant sources
- microbial sources
- bacterial
- fungi
34
common animal source enzymes: where are they sourced from?
- **lipase**
Animal pancreas
- **trypsin**
Ox bile
- **urokinase**
human plasma/cow urine
- **lysozyme**
milk/chicken albumen
- **adenosine deaminase**
bovine intestine
- **pepsin**
hog pancreas
- **dornase alpha**
recombinant human cells
35
plant sourced enzymes:
- **papain**
papaya
- **nattokinase**
natto bean
- **amylase**
barley
- **bromelain**
pineapple
36
bacterial enzymes:
- beta lactamase - staphylococci sp
- staphylokinase - staphylococci sp
- rhodanese - sulfobacillus sibiricus
- streptokinase - hemolytic streptococci
- l-aspariginase - E.coli
- collagenase - clostridium histolyticum
- amylase - bacillus sp.
37
bacterial enzymes:
- beta lactamase - staphylococci sp
- staphylokinase - staphylococci sp
- rhodanese - sulfobacillus sibiricus
- streptokinase - hemolytic streptococci
- l-aspariginase - E.coli
- collagenase - clostridium histolyticum
- amylase - bacillus sp.
38
what is the preferred source of enzymes? Why?
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
39
novel bio catalyst application:
- 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:
40
name two lysosomal storage diseases:
Fabry's disease
Gauchers disease
41
fabry's disease
- **galactosidase A [Gb3], used to break down what?**
used to break down a particular type of fat
- Deficiency in what
this is a disease due to a **deficiency** in the enzyme **alpha-galactosidase A**
- Causes a buildup in a fat called
**globotriaosylceramide** in the body - this is a complex lipid
42
Gauchers disease:
- **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
43
Oral and inhalable therapies:
- 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
44
cancer therapeutic enzymes:
L-asparaginase - oncolytic enzyme
45
Mechanism of action of L-asparaginase - oncolytic enzyme
- normal tissues synthesise L-Asparagine in sufficient quantities for protein synthesis
- neoplastic tissues require exogenous supply from circulation
46
L-aparaginase breaksdown circulating aa (L-asparagine) to what?
L-aspartate and ammonia which prevents protein synthesis of neoplasm → apoptosis
47
other important therapeutic enzymes:
48
What is the function of Lipase?
It breaks down fat.
49
Where can you find Lipase?
It is found in the pancreas, mouth and stomach.
50
How is Lipase usually sourced.
It is usually sourced from animal pancreas.
51
What is the function of Trypsin?
Helps to digest proteins by cleaving them.
52
Where can Trypsin be found?
It is found in the small intestine.
53
How is Trypsin sourced?
Ox bile
54
What is the function of Urokinase?
Converts inactive plasminogen into active plasmin.
55
Where is Urokinase located?
It is made in the kidney and found in urine
56
How is Urokinase sourced?
Human Plasma/Cow Urine
57
What is the function of Lysozyme?
It is an antimicrobial agent by cleaving the peptidoglycan components of bacterial cell walls which leads to cell death
58
Where is Lysozyme located?
Found in bodily secretions such as tears, saliva and milk
59
How is Lysozyme sourced?
Transgenic plants, animals and microorganisms that can produce human lysozyme
60
What is the function of Adenosine Deaminase?
It eliminated deoxyadenosine which is generated when DNA is broken down which is toxic to lymphocytes to deoxyinosine which is not harmful.
61
Where is Adenosine Deaminase located?
Produced in all cells however mostly in lymphocytes
62
How is Adenosine Deaminase sourced?
Bovine Intestine
63
What is the function of Pepsin?
Breaks down proteins in food during digestion
64
Where is Pepsin located?
Stomach
65
How is Pepsin sourced?
Hog pancreas
66
What is the function of Dornase a?
Is an inhaled medication that thins mucus. Reduces the number of lung infection and to improve lung function in patients with cystic fibrosis
67
Where is Dornase a located?
Medication
68
How is Dornase a sourced?
It is a highly purified solution of recombinant deoxyribonuclease I (rhDNase) an enzyme which actively cleaves DNA.
69
What is the function of Beta Lactamase?
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.
70
What is the function of Staphylokinase?
Activates plasminogen to form plasmin which digest fibrin clots.
71
What is the function of Rhodanese?
Main enzyme in sulfur metabolism for cyanide detoxification & anti-oxidative stress systems.
72
What is the function of Streptokinase?
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
What is the function of L-aspariginase?
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
What is the function of collagenase?
Break the peptide bonds in collagen. They assist in destroying extracellular structures in the pathogenesis of bacteria such as Clostridium.
75
What is the function of amylase?
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
What is the Principle behind Asparaginase?
Reduces the availability of asparagine, an amino acid that is necessary for the growth of some tumour cells.
77
What reaction does Asparaginase do?
Asparagine conversion
78
What are the therapeutic uses of Asparaginase?
Leukaemia
79
What is the Principle behind Collagenase?
Cleaves collagen
80
What reaction does Collagenase do?
Collagen hydrolysis
81
What are the therapeutic uses of Collagenase?
Skin ulcers
82
What is the principle behind Hyaluronidase?
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
What reaction is Hyaluronidase responsible for?
Hyaluronate hydrolysis
84
What are the therapeutic uses of Hyaluronidase
Heart Attack
85
What is the principle behind Ribonuclease?
Cleaves RNA
85
What is the principle behind Ribonuclease?
Cleaves RNA
86
What reaction is Ribonuclease responsible for?
RNA Hydrolysis
87
What is the therapeutic use of Ribonuclease?
Anti Viral
88
What is the principle behind Streptokinase?
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
What reaction is Streptokinase responsible for?
Plasminogen to Plasmin
90
What is the therapeutic use of Streptokinase?
Blood Clot
91
What is the principle behind Uricase?
Pegloticase, a recombinant mammalian uricase modified with monomethoxylpoly (ethylene glycol)(mPEG) is effective in treating refractory gout
92
What reaction is Uricase responsible for?
Urate to Allantoin
93
What is the therapeutic use of Uricase?
Gout
94
What is the principle behind Urokinase?
Urokinase-type plasminogen activator, is a serine protease present in humans and other animals.
95
What reaction is Urokinase responsible for?
Plasminogen to Plasmin
96
What is the therapeutic use of Urokinase?
Blood Clot
97
What is the principle behind Ribonuclease L?
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
What reaction is Ribonuclease L responsible for?
RNA Hydrolysis
99
What is the therapeutic use of Ribonuclease L?
Chronic Fatigue Syndrome
100
assaying for enzyme purity ( 2 tests)
- 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
what enzymes are measured in the plasma?
- plasma specific enzymes
- secreted enzymes
- cellular enzymes
102
when can enzymes be raised in the plasma?
- increased cell damage
- increased cell turnover
- proliferation of cells
- increased enzyme synthesis
- decreased clearance
103
what is the half life of an enzyme dependent on?
- the enzymes inactivation
- the enzymes removal
104
what determines the levels of enzymes in the plasma?
- degree of damage
- original intracellular levels
- amount of tissue that is affected
105
how can the enzymes original location be identified?
- measure organ specific enzymes
- measure isoenzymes
- analysis of patterns of several enzymes
106
organ specific enzyme examples:
- liver enzymes - transaminases (AST + ALT)
- pancreatic enzymes - pancreatic lipase + amylase
107
what are isoenzymes?
- catalyse the same reaction
- different primary structure
- different physical/ chemical properties
- differentiated
108
isoenzyme examples
Lactate Dehydrogenase
Creatine Kinase
109
What is the method used to measure lactate dehydrogenase?
Electrophoresis it gives you 5 different isoforms
110
Was is Creatine Kinase the same as?
creatine phosphokinase (CPK)
111
Creatine Kinase has 3 isoenzymes formed by combinations of different subunits what are they? Where are they abundant?
- 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
How do you detect CK iso-enzyme?
- electrophoretic separation
- column chromatography
113
How do you measure an isoenzyme? 6 different techniques
- electrophoresis
- inhibitor sensitives
- modified substrates
- coenzyme analogues
- thermostability
- immunoassay - this is if there is only slight differences between the isotypes
114
serum enzymes following myocardial infarction how does the graph look
115
enzyme profile for acute viral hepatitis graph explain
116
enzyme profile for acute alcoholic hepatitis explain graph
117
what is automation?
when a process is carried out by a device that is controlled by humans to perform it with a higher degree of efficiency
118
what is a semi-automatic device?
this is a device that is controlled by humans with a higher degree of human involvement than an automatic device
119
what are the benefits of automation? 7 benefits
- 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
how are automatic machines developed?
1. need for a method
2. method designed
3. increased throughput needed for the method
4. automated system developed
121
first instrument for enzyme analysis:
LKB
122
What is LKB?
an automated machine that has two different compartments
- one containing the substrate
- other containing buffers and substrates that are required for the reaction
123
samples are pipetted using what in a LKB machine?
a semi-automatic pipettor into a cuvette - on the bottom of the cuvette is a star shape groove
124
What happened to the cuvette containing the sample in a LKB machine?
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
What will happen when the sample reaches the nozzle?
it will then reach the nozzle for buffer and the correct amount will be added using automated syringes
126
Why is the substrate added?
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
Benefits of LKB
- there will be the same treatment for every sample
- reduced errors
- increased accuracy
- increased precision
128
Drawbacks of LKB
- 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
What has automation done?
- replaced all the manual steps - reduced manual labour
- walk-away capability - a technician isn't needed at all times
- increased reliability
130
What is a continuous flow analysis?
- 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
What is the concept behind continuous flow analysis?
- 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
How does the coil work in a continuous flow analysis?
gravity does the mixing within the coil
133
there are T-junctions added throughout the tube at locations where…
reagent might need to be added
134
The devices are limited by processing power. What is the rule?
the more processing power there is increases the throughput of the device
135
Why are automated analysers good? 5 points
- ideal for kinetic studies
- must be computer controlled
- discrete analysis
- disposable
- fast
136
What are the biological applications of automation?
- 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
What are the lag phase considerations of automation?
- 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
What is the automatic blanking consideration?
- 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
What are the common techniques for immobilisation of enzymes?
Absorption
Entrapment
Covalent Bonding
Cross Linking
140
What is adsorption?
- Physical binding of enzymes (or cells) on surface of an inert support
- Support may be inorganic or organic
141
What happens on inert support?
Involves weak forces such as Van der Waals forces and hydrogen bonds
142
How can the adsorbed enzymes be easily removed?
By minor changes in pH, ionic strength or temperature (this is a disadvantage for industrial use of enzymes)
143
Enzymes can be immobilised by physical entrapment inside what?How does this work?
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
Enzyme or cell not subjected to what?
Strong binding forces and structural distortions
145
How would some deactivation occur during immobilisation?
due to changes in pH/temperature/addition of solvents
146
What matrices are used for entrapping enzymes?
Polyacrylamide gel
collagen
gelatine
starch
cellulose
silicone
rubber
147
Where are the covalent bonds found?
Between the chemical groups of enzymes and the chemical groups of support
148
Disadvantage of covalent bonding
Loss of enzyme activity
149
Inert support requires what?
pre-treatment (to form pre-activated support) before it binds to enzyme
150
Cross-linking has an absence of what?
solid support
151
Enzyme molecules are immobilised how?
By creating cross links between them, through involvement of poly functional reagents
152
What do poly functional reagents do?
react with the enzyme molecules and create bridges which form the backbone to hold enzyme molecules
153
What is the most common poly functional reagent?
Glutaraldehyde
154
How does Glutaraldehyde reagent work?
React with lysyl residues of the enzymes and forms a Schiff’s base
155
Cross links formed between the enzyme and glutaraldehyde are…
irreversible and can withstand extreme pH and temperature
156
The cross links formed between the enzyme and glutaraldehyde are used to what?
immobilise several industrial enzymes
157
Advantages of glutaraldehyde + enzyme linkage
Simple
Cost effective
158
Disadvantages of glutaraldehyde + enzyme linkage
involves the risk of denaturation of the enzyme by the poly functional reagent
159
Lipase
Family of enzymes that break down triglycerides into free fatty acids and glycerol
160
Where is Lipase found?
In animal pancreas
161
What does Trypsin do?
Helps us digest protein
162
Where can Trypsin be found?
in the small intestine
(And Ox bile)
163
Trypsin breaks down what?
proteins, continuing the process of digestion that began in the stomach
164
Trypsin is a type of what enzyme?
proteolytic enzyme or proteinase
165
where is Trypsin produced?
in the pancreas
166
what is inactive Trypsin form called?
trypsinogen - produced in the pancreas
167
how does Trypsinogen enter the small intestine?
through the common bile duct and is converted into active trypsin
168
what does the active trypsin do?
acts with the other two digestive proteinases - pepsin and chymotrypsin
169
what happens after trypsin acts with the other two digestive proteinases - pepsin and chymotrypsin?
break down dietary protein into peptides and amino acids
170
what are amino acids essential for?
muscle growth, hormone production and other important bodily functions
171
Complications of inadequate trypsin levels
Malabsorption
Pancreatitis
172
Alanine transaminase (ALT) what reaction does it catalyse?
alanine + a-ketoglutarate → pyruvate + glutamate
173
what secondary reaction is used to determine the activity of the ALT enzyme?
lactate dehydrogenase reaction
pyruvate + NADH + H → lactate + NAD
174
what substrate has an absorbance that can be measured?
**NADH** - its consumption is equal to the production of pyruvate which is limited by the activity of the ALT enzyme
**@340nm**
175
how is the activity of an enzyme determined from the change in absorbance?
- it is determined using the molar extinction coefficient
**enzyme activity** (U/L) = **change in absorbance/min** X **molar extinction coefficient**
176
how is the coefficient of variance calculated?
CV= mean/SD
177
how is percentage coefficient of variance calculated?
CV (%)= mean/SD X 100
178
what does a low variance mean?
the data collected has high precision
