Week 11: Measurement Procedures in Chemistry

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Questions and Answers

What is the term used for the protein part of an enzyme?

Holoenzyme

Substrate

Cofactor

Apoenzyme (correct)

Which type of cofactor is typically derived from vitamins?

Prosthetic groups

Inorganic cofactors

Coenzymes (correct)

Activator

What is the role of the catalytic site in an enzyme?

It binds the substrate. (correct)

It prevents enzyme activity.

It serves as a regulatory site.

It modifies enzyme structure.

What distinguishes enzymes from other proteins?

Their ability to act as catalysts Signup and view all the answers

Which of the following correctly describes inorganic cofactors?

They are called activators. Signup and view all the answers

What is primarily measured to determine enzyme activity in a serum test?

Change in product concentration Signup and view all the answers

Which of the following conditions is NOT important in enzyme testing?

pH level of the substrate Signup and view all the answers

Why is it easier to measure catalytic activity rather than enzyme concentration?

Enzyme concentration is usually too low to measure accurately Signup and view all the answers

What are isoenzyme methodologies primarily used for?

To quantify enzyme concentration by mass Signup and view all the answers

Which of the following is a preferred method for testing enzyme activity?

Kinetic assay (Continuous monitoring method) Signup and view all the answers

What is the main purpose of a calibration curve in analytical chemistry?

To analyze the absorbance of unknown samples Signup and view all the answers

Which method relies on the known molar extinction coefficient?

Absolute Methods Signup and view all the answers

In a kinetic assay, what is measured during the substrate depletion phase?

Substrate consumption or product formation Signup and view all the answers

What characterizes a fixed-time kinetic assay?

Predetermined incubation time with absorbance measured at set intervals Signup and view all the answers

Which aspect is NOT part of an end-point assay?

Analysis of multiple data points continuously Signup and view all the answers

What is a critical requirement for continuous monitoring assays?

Multiple absorbance readings throughout the duration Signup and view all the answers

In kinetic assays, what type of assay tracks the absorbance in real-time?

Continuous Monitoring Assays Signup and view all the answers

Which of the following is NOT a characteristic of a kinetic assay?

Reagent is mixed and absorbance is measured post- incubation Signup and view all the answers

Which statement best describes the role of enzymes in biochemical reactions?

Enzymes act as catalysts and are not consumed. Signup and view all the answers

What distinguishes isoenzymes from regular enzymes?

Isoenzymes differ structurally but catalyze the same reactions. Signup and view all the answers

How does temperature affect enzyme activity?

Each enzyme has an optimal temperature for maximum activity. Signup and view all the answers

Which of the following is NOT a factor affecting enzyme activity?

Enzyme consumption in reaction Signup and view all the answers

What is the primary function of the Enzyme Commission (EC)?

To standardize enzyme nomenclature. Signup and view all the answers

What term describes enzymes that are controlled by genes and can lead to genetic diseases?

Enzyme systems Signup and view all the answers

What abbreviations are typically used to refer to clinically significant enzymes?

Common abbreviations unique to each enzyme. Signup and view all the answers

Which theory explains the formation of the substrate-enzyme complex?

Induced-fit theory. Signup and view all the answers

What is the primary role of an enzyme in a chemical reaction?

To accelerate the reaction without being consumed Signup and view all the answers

How do enzymes lower the activation energy of reactions?

By forming an enzyme-substrate complex Signup and view all the answers

Which of the following statements about enzymes is accurate?

Enzymes are usually specific to certain reactions Signup and view all the answers

What happens to the substrate during the enzymatic reaction?

It is converted into the product Signup and view all the answers

Which factor does NOT affect enzyme action?

Color of the substrate Signup and view all the answers

During which type of metabolic reaction do enzymes typically assist in breaking down compounds?

Catabolic reactions Signup and view all the answers

What role do R-groups in the active site of an enzyme play?

To help substrates bind to the active site Signup and view all the answers

How much substrate can a single maltase enzyme typically break down in a second?

1,000 molecules Signup and view all the answers

What happens to enzyme activity at temperatures above 50 ºC?

Activity is lost due to denaturation Signup and view all the answers

At what pH range do most physiologic enzymatic reactions occur?

7.0-8.0 Signup and view all the answers

What is the effect of increasing substrate concentration while enzyme concentration remains constant?

The reaction rate increases up to a maximum Signup and view all the answers

How does enzyme concentration affect the reaction rate when substrate concentration is in excess?

Reaction rate is proportional to enzyme concentration Signup and view all the answers

Which type of inhibitor binds to the active site of an enzyme?

Competitive inhibitor Signup and view all the answers

What is a common characteristic of enzyme inhibitors?

They cause a loss of catalytic activity Signup and view all the answers

What describes 'zero-order kinetics' in enzymatic reactions?

Rate is independent of enzyme concentration Signup and view all the answers

Which statement about enzyme denaturation is true?

It can be caused by physical or chemical factors Signup and view all the answers

Study Notes

Week 11: Measurement Procedures and Calculations

Clinical chemistry measurements use standardized procedures and calculations
Blank: A solution correcting for background interference, either from the sample or reagent.
Reagent blank: Used to avoid reagent interferences; contains all solution components except the patient sample.
Sample blank: Used to avoid sample interferences; contains all solution components except the principal reagent.
Standard: Pure solution with known concentration of the substance of interest, used as a reference point for comparing unknown concentrations.
Control: Solution with either normal or pathological known concentration of the substance of interest, used to check measurement accuracy.
Molar absorptivity: A constant for a given compound at a specific wavelength and path length, under specific conditions.
Transmittance: Ratio of transmitted to incident radiant energy.
Absorbance: Negative logarithm of transmittance (A = -logT).
Quantitative measurements: Necessary to standardize instruments before measuring intensity.

Objectives

Define terminologies associated with clinical chemistry measurements.
List fundamental solutions in spectrophotometric measurements.
Describe assay techniques in clinical chemistry measurements.
Discuss calibration curve.

Definition of Terms

Blank: A solution to correct background interference from the sample or reagent.
Standard: High-purity solution with a known concentration of a substance.
Control: Solution with a known concentration of a substance (normal or abnormal), used as a reference.

Quantitative Measurements

Standardizing of instruments is needed before intensity measurements.
Set minimum and maximum transmission conditions to give proper readings.
Maximum transmittance: Instrument set to give 100% transmittance using a blank.
Zero transmittance: Instrument set to give zero transmittance using a blank.

Methods for Calculations

Comparative Methods: Calculating unknown analyte concentration by comparing it to known standards or using a calibration curve.
Absolute Methods: Calculating unknown analyte concentration using known molar extinction coefficient.

Calibration Curve

Plotting absorbance on the Y-axis versus concentration on the X-axis using a series of standards of known concentration.
Used for analyzing concentration and verifying instrument function.

Assay Techniques in Clinical Chemistry

End-point assay: Reagent mixed with sample; absorbance measured after a specific incubation time.
Kinetic assay: Measuring substrate consumption or product formation between the end of the incubation period and substrate depletion phase. Two types: fixed time kinetic and continuous monitoring assay.
Fixed-time kinetic assay: Absorbance is measured at predetermined time intervals after a fixed incubation period.
Continuous monitoring assay: Absorbance readings are recorded continuously over the entire reaction time.

End-Point Example

Sample values for absorbance of controlled, standard and unknown solutions used and calculated to derive sample and control results.

Kinetic Examples

No examples provided in the slides.

Clinical Chemistry I: Week 12 - Enzyme Properties and Influencing Factors

Objectives: Defining enzymes and their structural components; discussing naming and classification.
Enzyme characteristics: Substrate-enzyme complex formation, kinetics comparison and clinically significant enzymes by name and abbreviation.
Enzyme activity measurement procedures: simplified procedure as well as fixed time and continuous monitoring assays, for measurement of enzyme activity.
Enzyme identification and determination of possible diagnoses based on chemistry test results.

Definition of the Enzyme

Large protein substances present in cells, act as catalysts (not consumed), highly specific for substrates.
Optimal conditions for enzyme activity include temperature and pH
Several thousand different enzymes in each cell, involved in multi-step biochemical pathways.
Regulated by feedback control and other mechanisms. .
Enzymes that catalyze the same reaction but differ in structure are known as Isoenzymes.

Enzymes Nomenclature

Standardized by the Enzyme Commission (EC), adding "ase" to the end (e.g., lactase) when the enzyme reacts with substrate.
Naming also includes recommended name, common abbreviation and standard abbreviation as well as EC code number and systemic name.

Enzyme Composition and Structure

Holoenzyme: The complete, active enzyme structure, consisting of apoenzyme and cofactors.
Apoenzyme: The protein component of the enzyme.
Cofactors: Non-protein components needed for enzyme activity (activators/coenzymes).
Catalytic site (active site): The region of the enzyme that binds the substrate.
Substrate: The substance upon which an enzyme acts in a chemical reaction
Allosteric site: A site on the enzyme other than the active site that can regulate enzyme activity.

Enzyme Cofactors

Non-protein substances bound to the protein portion of the enzyme (needed for maximal activity).
Two types of cofactors:
- Inorganic cofactors (activators): Such as chloride ions and magnesium ions.
- Organic cofactors (coenzymes): Small organic molecules derived from vitamins (e.g., NAD+, NADH, NADP+, NADPH).

Enzyme Reaction: Example (Illustrative Reactions)

Enzyme catalyzes a reaction involving a substrate and a cofactor to produce product I and product II.

Enzyme Catalytic Properties

Enzymes are catalysts (facilitate reactions without being consumed) catalyzing between 1 and 10000 substrate molecules per second.
Enzymes speed up chemical reactions by lowering the activation energy.

Enzymes are Catalysts

Lower the energy of activation by forming an enzyme-substrate complex.
Generally specific to reactions and substrates involved.
Active site: Pocket in the enzyme where the substrate binds during reactions.

Purpose of Enzymatic Reactions

Catabolism: Breaking down compounds.
Anabolism: Creating compounds.

How Enzymes Work (2 Theories)

Lock and Key Model: Enzyme active site is precisely shaped for one particular substrate to fit in the enzyme active site.
Induced Fit Model: Enzyme active site slightly adjust to shape the substrate for a better binding and catalysis.

Factors Affecting Enzyme Action:

Temperature: Optimum temperature maximizes reaction rate (usually around 37°C in humans); above that enzymes denature.
pH: Optimal pH range varies by enzyme, deviations can denature enzymes.
Substrate Concentration: Maximum activity is reached when all enzyme molecules are bound to substrate, the rate of reaction is directly proportional to the substrate concentration.
Enzyme Concentration: Reaction rate increases proportionally to enzyme concentration when there is enough substrate. Rate is dependent only on the concentration of the enzyme.
Inhibitors: Molecules that bind to the enzyme and reduce its activity; may be competitive, noncompetitive or uncompetitive, possibly affecting medication and drug design.

Testing of Enzymes

Enzymes are often markers of cell damage or disease.
Enzymes are present in small amounts but their activity can be measured easily in clinical labs to diagnose certain diseases.
Methods of measuring enzyme activity include fixed-time (end-point) and continuous monitoring (kinetic) methods.
Enzyme activity is calculated and reported in international units (IU)

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Description

This quiz covers standardized measurement procedures and calculations in clinical chemistry. Students will explore concepts such as blanks, standards, controls, molar absorptivity, and absorbance. Test your understanding of these essential measurement techniques and their applications in clinical settings.