MBG 311 - LDH Assay Presentation

LDH ENZYME KINETICS
MBG 311 Laboratory Week 9

Bilkent University
21.11.2023
WHAT IS AN ENZYME ?
• An enzyme is a substance that acts as a catalyst in living organisms, regulating the rate
at which chemical reactions proceed without itself being altered in the process
• Cell metabolism relies on enzymes to catalyze a broad spectrum of functions. This

involves the breakdown of food, with enzymes facilitating the transformation of
significant nutrient molecules (proteins, carbohydrates, and fats) into smaller, more
manageable compounds. Enzymes also contribute to the conservation and conversion
of chemical energy, as well as the assembly of cellular macromolecules using smaller
molecular precursors.
• Nearly all known enzymes are proteins, but RNA molecules called ribozymes can also
have catalytic activity.
HOW DO ENZYMES WORK ?
• They form enzyme-substrate complexes by binding the substrate through the active sites. This stabilizes
the transition state and lowers the activation energy, which in return speeds up the reaction.
• Some enzyme require cofactors to be active. Cofactors can be organic molecules (coenzymes) or inorganic
ions e.g., Ca2+ , Zn2+ (prosthetic group)
• The specificity of enzyme-substrate interactions is determined by the unique three-dimensional structure
of the active site.
• pH, enzyme concentration (transcription, translation, etc.), substrate concentration, temperature,
activators, and inhibitors can all regulate enzyme catalyzed reactions.
Fig 1: Sucrose hydrolysis by sucrase

ENZYME KINETICS AND MICHAELIS-MENTEN EQUATION
• Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes
• The concentration of substrate [S] is a key factor affecting the rate of a reaction catalyzed by an enzyme [E]
• The Michaelis-Menten equation describes the kinetic behavior of many enzymes
• V is the reaction rate at a substrate concentration [S]

• Vmax is the maximum rate that can be observed in the reaction.
• Km (Km = (k2 + k-1)/ k1 ) is the Michealis constant related to the
Graph 1: Effect of substrate concentration on the initial
velocity of an enzyme catalyzed reaction.
affinity of the enzyme for the substrate. Small Km means tight
binding, large Km means weak binding.
LACTATE DEHYDROGENASE
• Dehydrogenase is an enzyme that transfers a hydride from one molecule to another.

• LDH catalyzes the conversion of pyruvate (and NADH) to lactate (and NAD+) and back, in oxygen
absence and in liver (Cori cycle), respectively.
• LDH is extensively found in the body including blood cells, lungs, kidney, liver, muscles etc.
• LDH is released in the serum during tissue damage that’s why it is a common marker of tissue
injuries
LACTATE DEHYDROGENASE
• The conditions that lead to • Enzymatically active lactate dehydrogenase consists of four
increased LDH levels in the subunits (tetramer). They can form five possible tetramers
serum: (isoenzymes):
Ø Liver disease Ø LDH-1 : Present primarily in cardiac myocytes and
Ø Anemia erythrocytes.
Ø Heart attack Ø LDH-2 : Present mostly in white blood cells.
Ø Bone fractures Ø LDH-3 : Present in highest quantity in lung tissue
Ø Muscle trauma Ø LDH-4 : Highest amounts found in pancreas, kidney,
Ø Cancers
and placenta
Ø Infections (encephalitis,
meningitis, encephalitis, Ø LDH-5 : Highest amounts found in liver and skeletal
and HIV) muscle.
LDH ASSAY
• The aim of an LDH assay is to measure the activity of lactate dehydrogenase (LDH) in a sample
• LDH Kit component table:
Using Coenzyme 1 (R2) as the hydrogen carrier, LDH

catalyzes lactic acid to produce pyruvate. Pyruvate
reacts with 2, 4-dinitrophenylhydrazine (R3) and
forms pyruvate dinitrophenyhydrazon which gives
red- brown color in alkaline solution (R4). This color
can be measured using a spectrophotometer at 450
nm.
The kit also contains Pyruvic Acid with known

concentration used to generate the standard curve.
C ALCULATIONS
• Standard Curve: y = ax+b
• For Tissue Sample:
LDH Activity (U/L) = (ΔA450-b)/a*f/CPr
• ΔA450 : ODSample-ODControl
• f: Dilution factor of the sample before the test
• CPr: Concentration of protein in sample (mg/mL)
LDH ASSAY
• In our experiment we will be using liver sample. Concentration before dilution 9.3 mg/mL . Dilution factor 1:100
• We will measure the LDH activity of the sample over time (Generate a graph).
Standart Sample Control

duplicates duplicates duplicates
2h
1h 45 min
1h 30min
1h 15min
1h
45min
30min
15min

MBG 311 - LDH Assay Presentation

Uploaded by

Copyright:

Available Formats

MBG 311 - LDH Assay Presentation

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

MBG 311 - LDH Assay Presentation

Uploaded by

Copyright:

Available Formats

LDH ENZYME KINETICS

MBG 311 Laboratory Week 9

• Cell metabolism relies on enzymes to catalyze a broad spectrum of functions. This

Fig 1: Sucrose hydrolysis by sucrase

• V is the reaction rate at a substrate concentration [S]

• Dehydrogenase is an enzyme that transfers a hydride from one molecule to another.

• LDH Kit component table:

Using Coenzyme 1 (R2) as the hydrogen carrier, LDH

The kit also contains Pyruvic Acid with known

• Standard Curve: y = ax+b

• For Tissue Sample:

LDH Activity (U/L) = (ΔA450-b)/a*f/CPr

Standart Sample Control

You might also like