Clinical Chemistry I Week-12_Enzyme Properties and Reaction Influencing Factors PDF

Week 11 Measurement Procedures and Calculations 2 Objectives - Define terminologies associate with measurements in clinical chemistry - List fundamental solutions in spectrophotometric measurements - Describe assays techniques in clinical chemistry meas...

Week 11 Measurement Procedures and Calculations 2 Objectives - Define terminologies associate with measurements in clinical chemistry - List fundamental solutions in spectrophotometric measurements - Describe assays techniques in clinical chemistry measurements - Discuss about calibration curve Definition of Terms - Blank: A solution used to correct for background interferences that may come either from the sample or the reagent alone. I. Reagent blank: - A solution used to avoid interferences expected to come with reagents. - Contains all elements of the solution including the principal regent, buffer solution and others except the patient sample. II. Sample blank: - A solution used to avoid interferences expect to come with samples. - Contains all elements of the solution including the patient sample, buffer solution and others except the principal regent. Definition of Terms - Standard: - Pure solution having known concentration of interested substance in a fixed value (Single number). - Used as a reference value to compare the concentration of standard solution. - Control: - Matrix solution having either normal or pathologic known concentration of interested substance in a defined range. - Used to control the over all activities of the measurement. - Normal and Pathologic (Depends on the analyte: Low, High or Both) Definition of Terms - Molar absorptivity: A constant for one molar solution of a given compound at a given wavelength and a 1-cm path length under prescribed conditions of solvent, temperature, pH. - Transmittance: A ratio of transmitted radiant energy to incident radiant energy. - Absorbance: Negative logarithm of transmittance. Quantitative Measurements - Necessary to standardize instrument before measurement of intensity. - The basic procedure involves setting the minimum & maximum conditions of transmission & adjusting the metering to give appropriate readings - Maximum transmittance: Blank sample is used & instrument is adjusted to give either a reading of 100% transmittance or zero absorbance. - Zero transmittance: All light to the detector is cut off & the meter is adjusted to give a transmittance reading of zero. 7  Methods for Calculations There are two methods for calculations of analyte concentration of an unknown solution: I. Comparative Methods - Ratio of known standard to unknown - Use of standard calibration curve II. Absolute Methods - Use of known molar extinction coefficient 01-Dec-24 8  Calibration Curve Calibration can be prepared by plotting absorbance on the Y-axis versus concentration on the X-axis coordinate graph paper for a series of standard solution of known concentration Application of calibration curve: I. Analysis of concentration II. Verifying the proper functioning of analytical instruments 01-Dec-24 9 Assay Techniques in Clinical Chemistry Basically there are different types of analytical techniques: I. End-point Assay II. Kinetic Assay: A. Fixed Time Kinetic B. Continuous Monitoring End-point Assay - Reagent mixed with sample, and Abs is measured after incubation for time interval. - The reaction take place (Different reaction types: Enzymatic, Binding). - Absorbance reading at any time after incubation, and during the result reading time - Analyte concentration calculated by using appropriate absolute or competitive method End-Point Example 12 Kinetic Assay - Kinetic assay is a method in which the amount of substrate consumption or product formation is measured between end of incubation period, and substrate depletion phase. - Increasing or Decreasing - Two types of kinetic: I. Fixed-Time Kinetic II. Continuous Monitoring Assay (3 data points at least) Kinetic Assay - Fixed-Time Assay: Substrate is added and Abs is measured after a predetermined incubation period, at fixed time intervals (Starting point and Ending point). - Continuous Monitoring Assay: Multiple absorbance readings are recorded continuously over the entire reading time interval. Kinetic Examples 15 See YOU Next Lecture  Clinical Chemistry I Medical Laboratory Sciences 0202304 Department Week-12_Enzyme Properties and Reaction Influencing Factors Textbook: Instructor: Mohammad QABAJAH - Rifai, N., Horvath, A.R., & Wittwer, C.T. (2019). Tietz Fundamentals of E-mail: [email protected] Clinical Chemistry and Molecular Diagnostics (8th ed.) Objectives 2 I. Define the enzyme and its structural components II. Discuss enzyme characteristics naming and classification III. Explain how enzymes are working and the factors affecting their activity IV. Compare the two theories of Substrate-Enzyme complex formation V. Differentiate first-order kinetics and zero-order kinetics VI. List the clinically significant enzymes by name and abbreviation VII. Explain how enzymes are commonly tested (Fixed time and continuous monitoring assays) VIII. Discuss the simplified procedure for the measurement of enzyme activity IX. Given chemistry test results, identify abnormal results (low, normal, high) and give possible diagnosis based on results Serum Enzymes Definition of the Enzyme 3 Definition of the Enzyme 4 - Large protein substances present in the cell in small amounts; Act as "catalysts“ (Not consumed) - Are highly specific for their substrate - Each enzyme shows optimal conditions for activity: Temperature, pH, etc. - 2,000 to 3,000 different enzymes in each cell (Different enzyme set/cell type) - Involved in multi-step biochemical pathways - Subject to regulation by feedback control and other mechanisms - Isoenzymes (Isozymes): Enzymes that catalyze the same reactions but differ structurally - Production directly controlled by genes; Many "genetic diseases" causing enzyme system defects Enzymes Nomenclature 5 - Standardized by the Enzyme Commission (EC) of the International Union of Biochemistry (IUB) - Adding “ase” on the end (e.g. Lactase): When it reacts with substrate - Adding “in” on the end (e.g. Pepsin): When is digest the compound - Each enzyme has a: - Recommended Name – e.g. Lactate Dehydrogenase - Common Abbreviation – e.g. LDH - Standard Abbreviation – e.g. LD - EC code Number – e.g. 1.1.1.27 - Systemic Name – e.g. L-Lactate: NAD+ oxidoreductase Enzyme Composition and Structure 6 - Enzymes are distinguished from other proteins by their catalytic activity - Any structure changes in can affect the catalytic activity of the enzyme - Structural Component s I. Holoenzyme: - Apoenzyme: Protein part of the enzyme - Cofactors: Activator or coenzyme; Non-protein substance II. Catalytic Site (Active Site): - Is the region of an enzyme that binds the substrate III. Substrate: - Substance upon which an enzyme acts on in a chemical reaction IV. Allosteric Site: - A cavity of the enzyme other than the active site (e.g. Regulatory site) Enzyme Cofactors 7 Non-protein substances bound to the protein portion of the enzyme: needed for maximal activity Two Types of cof actors: I. Inorganic cofactors are called activators - Such as chloride ions and magnesium ions II. Organic cofactors are called coenzymes - Coenzymes are small organic molecules which are derived from vitamins - Nictotinamide adenine dinucleotide (NAD), NADH, NADP and NADPH - Organic cofactors tightly bound to the enzyme are called Prosthetic group Enzyme Reaction: Example 8 Enzyme Substrate Cofactor Product I Product II (Coenzyme) Serum Enzymes Enzyme Catalytic Properties - Enzymes are Catalysts - How Enzymes Work? - Factors Affecting Enzyme Action 9 Enzymes are Catalysts 10 - A catalyst is any substance which makes a chemical reaction go faster, without being changed or consumed - An enzyme generally can typically catalyze between 1 and 10,000 molecules of substrate per second - Enzymes speed up reactions by lowering the activation energy of reactions, which is the energy must be supplied in order for molecules to react with each others Enzymes are Catalysts 11 - Enzymes lower the energy of activation by forming an enzyme-substrate complex - General relationship among enzyme, substrate and product - Enzymes are usually specific as to the reactions they catalyze and the substrates that are involved in these reactions - Active site: - Remember, Protein folding Purpose of Enzymatic Reactions 12 Metabolism: Organization the chemical reactions in a cell - Catabolic reactions: Compounds are broken down - Anabolic reactions: Compounds are synthesized For example, - Maltose is made of two glucose molecules - The maltase enzyme is a protein is specifically shaped to accept a maltose molecule and break the bond - The two glucose molecules are released - A single maltase enzyme can break ~1,000 bonds/sec  How Do Enzymes Work? 2 theories 13 - Enzyme binds a substrate in the active site - Enzyme structure flexible, not rigid - Only certain substrates can fit the active site - Enzyme and active site adjust shape to bind - R-groups in the active site help substrate to bind - Increases range of substrate specificity - Enzyme-substrate complex forms - Shape changes improve catalysis during reaction - Substrate reacts to form product - Product is released Factors Affecting Enzyme Action 14 I. Temperature II. pH III. Substrate Concentration IV. Enzyme Concentration V. Inhibitors Factors Affecting Enzyme Action: Temperature 15 Temperature - Little activity at low temperature; Rate increases with temperature - Most active at optimum temperatures, close to that of the physiologic environment of enzyme - Usually 37°C in humans - Activity lost with Denaturation at high temperatures (40-50 ºC) Denaturation: - Causes change in enzyme structure: Loss of activity - Caused by: Elevated temperature; extreme change in pH and certain chemicals Factors Affecting Enzyme Action: pH 16 - Maximum activity at optimum pH - Most physiologic enzymatic reactions occur in the pH range of 7.0-8.0 - Narrow range of activity - Most lose activity (denature) in low or high pH Factors Affecting Enzyme Action: Substrate Conc. 17 - Increasing substrate concentration increases the rate of reaction (if enzyme conc. is constant) - Maximum activity reached when all of enzyme combines with substrate - Reaction rate is directly proportional to substrate concentration (First-order kinetics) - Dependent on substrate Factors Affecting Enzyme Action: Enzyme Conc. 18 - As long as the substrate concentration exceeds the enzyme concentration, the speed of the reaction is proportional to the enzyme concentration - Rate of reaction is dependent on the concentration of the enzyme only - Occurs only when there is an excess of substrate. This is zero-order kinetics - Independent of substrate Factors Affecting Enzyme Action: Enzyme Inhibitors 19 - Cause a loss of catalytic activity - Change the protein structure of an enzyme - Some effects are irreversible - May be - Competitive: Inhibitor binds to active site - Noncompetitive: Inhibitor binds to site other than active site - Uncompetitive: Inhibitor binds to ES complex - Reality: Usef ul in medication and drug designing Serum Enzymes Testing of Enzymes 20 Testing of Enzymes 21 WHY to test enzymes? - Enzyme itself is not a health problem instead it is a marker of cell death - The enzymes are synthesized in cells and remain there (unless specifically secreted by the cells), except when the cells are turning over (natural cell cycle) or are damaged - Enzymes are markers of disease; useful in diagnosis of particular diseases - Enzymes are present in small amount s in fluids - Difficult to isolate and measure the enzyme itself (concentration) - Easier to measure catalytic activity: Activity is relative to concentration Testing of Enzymes: Measurement of Activity 22 Procedure for simplifi ed enzyme test : Enzyme activity based on the reaction it catalyzes I. Enzyme present in the serum; (Specimen: Serum) II. Add substrate (present in reagent) to start the reaction. Remember (Optimal Conditions) III. Incubate in 37ºC incubator IV. Measuring: Common is photometrically (Spectrophotometer: Continuous method) - Increase in product concentration - Decrease in substrate concentration - Decrease in coenzyme concentration - Increase in concentration of altered coenzyme V. This is proportional to enzyme present; Perform calculation to get enzyme result Testing of Enzymes: Measurement of Activity 23 Main approaches to test ing enzymes; both are based on enzymatic reactions I. Fixed time method (End-point assay) II. Continuous monitoring method (Kinetic assay); preferred method Isoenzyme methodology is different: 2 common techniques I. Immunoassay methodologies that quantify enzyme concentration by mass (e.g. CK-MB) II. Electrophoresis especially for isoforms and isoenzymes Measurement of Activity: Fixed-Time Method 24 Fixed-Time Method (End-point) - Reactants are combined - Reaction proceeds for a designated amount of time - Reaction stopped: Inactivating enzyme with weak acid - Measurement is made of the amount of the reaction that has occurred - 1 Measurement taken - Disadvantage - Will not notice deviations from linearity Measurement of Activity: Continuous Monitoring 25 Cont inuous Monitoring Method (Kinet ic): Method of Choice - Reactants are combined - Reaction proceeds while multiple measurement are taken while reaction occurs - Either at specific time interval (usually every 30 or 60 seconds) - Or continuously by a continuous recording spectrophotometer Measurement of Activity: Activity Calculation 26 Calculation of Enzyme Activity - EC says to report enzymes in international unit s (IU) IU is defined as the amount of enzyme that will catalyze the reaction of 1μmol of substrate per minute under specified condition of temperature, pH, substrates and activators (Cofactors) - Usually enzyme concentration is express in units per liter (IU/L or U/L) Enzymes as Reagents 27 Enzymes may be used as reagents to measure many nonenzymatic constituents in serum - Example glucose, cholesterol Remember the enzymatic reactions for glucose (e.g. Hexokinase): If substrate is present in serum (glucose) by adding the enzyme hexokinase (present in the reagent) you are starting the enzymatic reaction Serum Enzymes Enzymes of Clinical Significance 28 Clinically Significant Enzymes 29 List of Clinically Significant Enzymes - Liver Enzymes: - Aspartate Aminotransferase - Alanine Aminotransferase - Alkaline Phosphatase - Gamma Glutamyltransferase - Cardiac Enzymes: - Creatine Kinase - Lactate Dehydrogenase - Amylase - Lipase - Acid Phosphatase - Glucose-6-Phosphate Dehydrogenase - Cholinesterase  30 See YOU Next Lecture