Laboratory method for measuring enzyme activity.
Vital for study of enzyme kinetics and enzyme inhibition.
Measurement of enzyme activity – follow the change in concentration of substrate or product – measure reaction rate.
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.com
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.com
The flux of metabolites through metabolic pathways involves
catalysis by numerous enzymes. Active control of homeostasis is achieved by the regulation of only a small number of enzymes.
This ppt describes the overview of enzyme regulation and Allosterism. Presented since October 23,2017GC at Addis Ababa University, School of Medicine, Department of medical biochemistry.
Some of the enzyme possess additional sites, known as allosteric sites besides the active site . Such as know as allosteric enzyme. The allosteric sites are unique place on the enzyme molecules allosteric enzyme have one or more allosteric site.
HISTRY
The term allosteric has been introduced by the two Noble Laureates JACOB AND MONOD to denote an enzyme site different from the active site which non competitively bands molecule other than the substrate and may influence the enzyme activity.
Properties of allosteric enzyme
Effector may be positive or negative, this effector regulate the enzyme activity . The enzyme activity is increased when a positive allosteric effector binds at the allosteric site known as activator site. On the other hand negative allosteric effector bind at the allosteric site called inhibitor site and inhibit the enzyme activity
The flux of metabolites through metabolic pathways involves
catalysis by numerous enzymes. Active control of homeostasis is achieved by the regulation of only a small number of enzymes.
This ppt describes the overview of enzyme regulation and Allosterism. Presented since October 23,2017GC at Addis Ababa University, School of Medicine, Department of medical biochemistry.
Some of the enzyme possess additional sites, known as allosteric sites besides the active site . Such as know as allosteric enzyme. The allosteric sites are unique place on the enzyme molecules allosteric enzyme have one or more allosteric site.
HISTRY
The term allosteric has been introduced by the two Noble Laureates JACOB AND MONOD to denote an enzyme site different from the active site which non competitively bands molecule other than the substrate and may influence the enzyme activity.
Properties of allosteric enzyme
Effector may be positive or negative, this effector regulate the enzyme activity . The enzyme activity is increased when a positive allosteric effector binds at the allosteric site known as activator site. On the other hand negative allosteric effector bind at the allosteric site called inhibitor site and inhibit the enzyme activity
Enzyme inhibition is explained with its kinetics, animations showing mechanism of inhibitors action, examples of inhibitors are explained in detail with Enzyme inhibited.
by Dr. N. Sivaranjani, MD
DIURETICS
Diuertics are the drugs used to increase the urine output by excretion of Na+ and water from the kidney.
Primary effect: Reduce absorption of sodium and chlorine ions from the filtrate.
Secondary effect: Increased water loss along with the excretion of sodium and chlorine.
CLASSIFICATION
Based on mechanism of action and site of action
Acting on PCT
a. Carbonic anhydrase inhibitor
Acetazolamide
Dorazolamide
Metazolamide
b. Xanthine derivative
Aminophylline
Theophylline
2. Act on loop of Henlee
a. Osmotic Diuretic
Mannitol
Glycerin
Urea
b. Loop diuretic/ High ceiling
Furosemide
Torsemide
Ethacrynic acid
3. Drug acting on DCT
a. Thaizide diuretic
Chlorthiazide
Hydrochlorthiazide
Hydroflumethazide
Bendroflumethazide
Benzthiazide
Cyclopenthiazide
b. Thiazide like diuretic
Chlorthalidone
Indapamide
Metolazine
4. Drugs acting on collecting duct
a. Aldosterone antagonist
Spironolactone
b. Directly acting
Amiloride
Triamterine
Major application of diuretics ;
Used in congestive heart failure
Essential hypertension
Acute and chronic heart failure
Currently used screening methods are based on effect of drug on water and electrolyte metabolism in rats.
SCREENING METHODS
IN VIVO METHODS :
Diuretic activity in rats [LIPSCHITZ TEST]
Saluretic and diuretic activity in dogs
Saluretic activity in rats
Clearance methods
Stop flow technique
Micro puncture technique in rat.
IN VITRO METHODS :
Carbonic anhydrase inhibition in vitro
Patch clamp technique in kidney cells
Isolated perfused kidney
Perfusion of isolated kidney tubules
1. CARBONIC ANHYDRASE INHIBITION IN-VITRO
PURPOSE AND RATIONALE
Carbonic anhydrase is a Zn containing enzyme.
H2CO3 H20+CO2
Inhibition of CARBONIC anhydrase in PCT causes
● Decreased H+ ion formation
● Decreased Na+/H+ antiport
●Increased Na+and HCO3- in lumen
●increased excretion of Na+HCO3-
●Increased production of alkaline urine
PROCEDURE
The analytical method is based on the catalysis of the conversion of CO2 to H2CO3 by the enzyme , with resulting decrease in pH being monitored colorimetrically.
ASSAY
■ CO2 flow rate is adjusted to 30 to 45 ml/min
■ 400 µl phenol red indicator solution
■ 100µ l enzyme.
■ 200 µl H2O or appropriate drug concentration after 3min of equilibriation.
■ 100 µl carbonate/bicarbonate buffer is added.
■ The following parameters are determined in duplicate samples :
Tu = [uncatalysed time]=time for the colour change to occur in the absence of enzyme.
Te =[Catalysed time]=time for the colour change to occur in presence of enzyme.
Tu – Te = enzyme rate
Ti = enzyme rate in the presence of various concentrations of inhibitor.
EVALUATION
Percentage inhibition of carbonic anhydrase is evaluated
% evaluation =1
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Ores recovered by mining include metals, coal, oil shale, gemstones, limestone, dimension stone, rock salt, potash, gravel, and clay. Mining is required to obtain any material that cannot be grown through agricultural processes, or created artificially in a laboratory or factory. Mining in a wider sense includes extraction of any non-renewable resource such as petroleum, natural gas, or even water.
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if your doing fish dissection and need some anatomical information then go through my slides.
in this i have written fish anatomy with its physiological implications
Are you looking for some good journals to publish your data? Then this is the correct time to read my article. I am writing this with the hope of inhibiting you from publishing your data in predatory and fake journals.
These are the following criteria you should know before submitting your manuscript to a journal:
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Secondary structure prediction has been around for almost a quarter of a century. The early methods suffered from a lack of data. Predictions were performed on single sequences rather than families of homologous sequences, and there were relatively few known 3D structures from which to derive parameters. Probably the most famous early methods are those of Chou & Fasman, Garnier, Osguthorbe & Robson (GOR) and Lim. Although the authors originally claimed quite high accuracies (70-80 %), under careful examination, the methods were shown to be only between 56 and 60% accurate (see Kabsch & Sander, 1984 given below). An early problem in secondary structure prediction had been the inclusion of structures used to derive parameters in the set of structures used to assess the accuracy of the method.
Some good references on the subject:
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
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holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
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Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
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Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
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IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
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Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
2. • Laboratory method for measuring enzyme
activity.
• Vital for study of enzyme kinetics and enzyme
inhibition.
• Measurement of enzyme activity – follow the
change in concentration of substrate or
product – measure reaction rate.
3.
4. DIRECT CONTINUOUS ASSAYS
• Difference in properties of substrate and product –
measured directly.
• Continuous observation of the progress curve – most
preferred.
• Change in
– Absorbance. - Fluorescence.
– pH. - Optical rotation.
– Enthalpy. - Viscosity.
– Volume of reaction mixture.
5. ABSORBANCE
◦ I - intensity of light at a specified wavelength passing through aλ
sample.
◦ I0 - intensity of the light before it enters the sample.
Relation between concentration and absorbance:
◦ extinction – proportionality constant relating absorbance toɛ →
concentration.
◦ c – concentration.
−=
0
10log
I
I
A
cIA =∈
∈
=
A
c
6. TURBIDIMETRY
• Light scattering not absorbance.
• Action of enzymes on turbid polymer solutions.
• Difficult to standardize – difficult to reproduce
results.
• E.g.: bacterial lysozyme assay – on dried bacterial
cells – measured at 450nm.
• Unit: one unit of activity – initial rate of change in
absorbance of 0.001 per minute when the volume in
the cuvette is 2.6ml, pH- 6.24 at 25˚C.
7. FLUORESCENCE
• Result of electronic transition – converts the
absorbing molecule to an excited state.
• Fluorescent molecule emits part of absorbed
energy as light – lower energy but higher
wavelength.
• More sensitive than absorbance assays.
8. FLUORIMETRY
• Fluorospectrophotometer – more specific than
spectrophotometer.
• Disadvantage: fluorescing molecules quench
in solution.
• E.g: anthranilate synthase
Chorismate + L-glutamine anthranilate + pyruvate↔
• λexci = 325nm, λemi = 400nm.
9. RADIOMETRY
• Requirement of labelled substrates and counting
instruments.
• Substrates can be labelled with 14
C, 3
H, 32
P, 35
S, 125
I.
• E.g: galactosyl transferase.
UDP-galactose* + glucosamine UDP + lactosamine*
• Stop the reaction by adding EDTA.
↓
Pass through ion exchange column – separate substrate and
products.
↓
Product collected – check radioactivity by scintillation counter.
GT, Mn+2
10. pH stat
Stationary pH.
Used to monitor progress of chemical reaction in
which protons are liberated or taken up.
Achieved by measuring the amount of acid or base
required to be added to maintain constant pH.
11. DIRECT DISCONTINUOUS ASSAY
p-nitrophenol in
alkaline condition
– highly
electronegative.
Colorless in acidic
condition and
yellow in alkaline
condition.
Yellow color
measured at
405nm.
12. INDIRECT ASSAYS
• Further treatment of reaction mixture –
produce a measurable product or increase
sensitivity of assay procedure.
13. CONTINUOUS ASSAYS
• Manipulation necessary to detect product formation – allows
continuous observation of the change.
• Less prone to errors from sample manipulation in
discontinuous assays
• Reagents required for color development or measurement of
activity included in the reaction mixture.
• E.g.: carnitine acyl transferase.
Acyl CoA + carnitine acyl carnitine + CoASH↔
CoASH + 5,5’-dithiobis-2-nitrobenzoate 4-nitrothiolate anion→
(DTNB - reagent)
• λmax = 412nm.
14. DISCONTINUOUS ASSAYS
• Also called sampling assay.
• Stopping reaction - after a fixed time.
• Treating the reaction mixture to separate the product for
analysis or produce a measurable change in properties of
substrates or product.
• Separate product for analysis (radiochemical assay)
– No modification made on the substrate/product can be considered→
as a direct assay.
• Produce change in properties of one substrate/product can→
be measured.
– Formation of ATP can be determined by measuring light intensity in
the presence of luciferase.
ATP + luciferin +O2 oxyluciferin + PPi +CO→ 2 + AMP + light
15. Coupled assays
• Use of one or more additional enzymes to
catalyse a reaction of one of the products to
yield a compound that can be directly
detected.
• Additional enzyme – coupled enzymes.
16. Examples
• Hexokinase.
– Coupling of the
formation of
glucose-6-
phosphate to the
reduction of
NADP+
in the
presence of G6P
dehydrogenase.
Glucose
ATP, Mg2+
ADP, Mg2+
Glucose 6-phosphate
NADP+
NADPH + H+
6-Phosphogluconolactone
G6P
DEHYDROGENASE
HEXOKINASE
19. 2. Alanine aminotransferase (serum glutamate pyruvate
transaminase)
• ALT/SGPT.
Alanine + α-ketoglutarate pyruvate + glutamate↔
Pyruvate + NADH + H+
lactate + NAD↔ +
3. Decarboxylase.
Lysine cadaverine + CO2
CO2 + PEP oxaloacetate
Oxaloacetate + NADH + H+
malate + NAD+
Lysine decarboxylase
Wheat PEP carboxylase
MDH
20. Validity of results
• Reaction step should not be rate limiting.
• Velocity of the reaction increases till coupling
enzyme reaches the rate of the first enzyme.
• Coupling enzyme – high Km for the enzyme
and low Km for substrate.
22. Forward coupled assay
• Malate dehydrogenase.
Malate + NAD+ MDH Oxaloacetate + NADH + H+
Acetyl CoA
CoA
CITRATE SYNTHASE
Citrate
23. References
• Enzyme Assays by Robert Eisenthal.
• Photometric assays – Robert A. John.
• Principles of enzyme assays and kinetic
studies – Keith F. Tipton.
Editor's Notes
Photometric assays – Robert A. John.
Photometric assays – Robert A. John
Photometric assays – Robert A. John
Principles of enzyme assays and kinetic studies – Keith F. Tipton.
Enzyme Assays - Robert Eisenthal and Michael J. Danson
Principles of enzyme assay and kinetic studies – Keith F. Tipton