Measuring Enzyme Activity Lecture Notes PDF

Introduction to Medical Sciences 1 Building Blocks of Cells part 2 Measuring enzyme activity Copyright © Brunel University London v.3 2024. All rights reserved Building Blocks of Cells Dr Ricardo Carnicer Hijazo 2024 Version 3 [email protected]...

Introduction to Medical Sciences 1 Building Blocks of Cells part 2 Measuring enzyme activity Copyright © Brunel University London v.3 2024. All rights reserved Building Blocks of Cells Dr Ricardo Carnicer Hijazo 2024 Version 3 [email protected] Copyright © Brunel University London v.3 2024. All rights reserved 5  Properties of enzymes The enzyme only reacts with a few Substrate closely related Products substrates The enzyme only reacts with a few Active site closely related substrates Enzyme Cofactor The enzyme is not The enzyme is not consumed during the consumed during Image created by TimVickers. Wikipedia public domain. reaction the reaction Copyright © Brunel University London v.3 2024. All rights reserved 6 How to measure enzyme activity? Enzyme activity: Amount of product that is produced per unit of time. Substrate T = 1 min Product Enzyme 2 mol / minute Diagram created by RCH. Public Domain Copyright © Brunel University London v.3 2024. All rights reserved 7  How to measure enzyme activity? Enzyme activity: Amount of product that is produced per unit of time. Substrate T = 1 min Product Enzyme Spectrophotometric enzyme assays: A spectrophotometer is a piece of equipment that measures the absorbance of light of the product/substrate during the enzymatic reaction. Copyright © Brunel University London v.3 2024. All rights reserved 8 Spectrophotometer Abs = 0.021 Lamp Detector Diagram created by RCH. Public Domain Absorbance increases as a compound accumulates in the cuvette. Copyright © Brunel University London v.3 2024. All rights reserved 9 Spectrophotometer: Absorbance from the products per unit of time. Abs = 0.021 Lamp Detector Enzyme activity: Amount of product that is produced per unit of time. Copyright © Brunel University London v.3 2024. All rights reserved Beer-Lambert Law: Calculation of concentration Absorbance is directly proportional to the concentration of the compound. A=ExCxℓ A = Absorbance E = Coefficient of absorption C = Concentration of compound L = Path of light through the cuvette TBL 2_Building Blocks of Life, Cells, Organelles Copyright © Brunel University London v.3 2024. All rights reserved 11 Beer-Lambert Law: Calculation of concentration Absorbance is directly proportional to the concentration of the compound. A CA = = ExCxℓ Exℓ A = Absorbance E = Coefficient of absorption C = Concentration of compound L = Path of light through the cuvette TBL 2_Building Blocks of Life, Cells, Organelles Copyright © Brunel University London v.3 2024. All rights reserved 12  Tube 0 1 2 3 4 5 6 0 0.1 0.2 0.3 0.4 0.5 0.6 Substrate, mM Diagram created by RCH. Public Domain Enzyme + Cofactors + Substrate Copyright © Brunel University London v.3 2024. All rights reserved 13  Tube 0 1 2 3 4 5 6 0 0.1 0.2 0.3 0.4 0.5 0.6 Substrate, mM Absorbance Copyright © Brunel University London v.3 2024. All rights reserved 14  Tube 0 1 2 3 4 5 6 0 0.1 0.2 0.3 0.4 0.5 0.6 Substrate, mM Absorbance Concentration Copyright © Brunel University London v.3 2024. All rights reserved 15  0 1 2 3 4 5 6 Tube 0 0.1 0.2 0.3 0.4 0.5 0.6 Diagram created by RCH. Public Substrate, mM Domain Absorbance Concentration Velocity = concentration/time Tube 0 1 2 3 4 5 6 Substrate, mM 0 0.1 0.2 0.3 0.4 0.5 0.6 Velocity, µM/min 0 16 22 24 27 29 30 Copyright © Brunel University London v.3 2024. All rights reserved 16 Tube 0 1 2 3 4 5 6 Substrate, mM 0 0.1 0.2 0.3 0.4 0.5 0.6 Velocity, µM/min 0 16 22 24 27 29 30 Velocity of product formation 40 30 20 10 0 Substrate, mM Copyright © Brunel University London v.3 2024. All rights reserved 17 Tube 0 1 2 3 4 5 6 Substrate, mM 0 0.1 0.2 0.3 0.4 0.5 0.6 Velocity, µM/min 0 16 22 24 27 29 30 Velocity of product formation 40 30 20 10 0 Substrate, mM Copyright © Brunel University London v.3 2024. All rights reserved 18 Importance of Clinical Enzymology Clinical enzymology studies the activity and properties of enzymes in samples (usually of blood) taken from patients. Enzymes are use in the diagnosis of diseases. Example: Enzymes commonly studied for diagnosis of liver diseases are: 1. Alanine amino transferase (ALT) 2. Alkaline phosphatase (ALP) 3. Nucleotide phosphatase (NTP) 4. Gamma glutamyl transferase (GGT) Copyright © Brunel University London v.3 2024. All rights reserved 19 To contact me: [email protected] Copyright © Brunel University London v.3 2024. All rights reserved