Biology 201 Final Prep PDF

Lab #2: PHOTOMETRY 1. Absorption and Color Perception ➔​ When white light (containing all wavelengths in the visible spectrum) passes through a solution containing a coloured compound, the compound's atoms or molecules absorb specific wavelengths. ➔​ The wavelengths that are not...

Lab #2: PHOTOMETRY 1. Absorption and Color Perception ➔​ When white light (containing all wavelengths in the visible spectrum) passes through a solution containing a coloured compound, the compound's atoms or molecules absorb specific wavelengths. ➔​ The wavelengths that are not absorbed are transmitted through the solution and are perceived as the compound's colour. ➔​ Example: Riboflavin ◆​ Riboflavin absorbs light primarily in the blue region of the visible spectrum, with a maximum absorption at 450 nm. ◆​ This absorption removes blue light from the white light, leaving the other wavelengths (primarily red and green) to be transmitted. These combine to appear yellow to the human eye. ◆​ Riboflavin also absorbs ultraviolet (UV) light at 260 nm and 370 nm, but since these wavelengths are outside the visible range (400–700 nm), they do not affect the perceived colour. 2. Photometry ➔​ Definition: Photometry is the measurement of light absorption by compounds. It uses the interaction of light with matter to analyze substances qualitatively (what is present) and quantitatively (how much is present). ➔​ The absorption spectrum extends across different regions of the electromagnetic spectrum: ◆​ UV region: 200–400 nm ◆​ Visible region: 400–700 nm ◆​ Near-infrared (IR) region: 700–900 nm 3. Lambert's Law ➔​ Description: Lambert's law states that light absorption is: ◆​ Independent of the intensity of the incident light. ◆​ Proportional to the thickness of the absorbing medium (solution). ➔​ In practical terms, each layer of the solution absorbs the same fraction of light that enters it. This principle is fundamental to determining the concentration of a compound in a solution using photometric techniques. Beer's Law ​ Statement: The light absorption by a solution is directly proportional to the number of absorbing molecules in the light's path.  ​ Implication: If the absorbing substance is dissolved in a transparent solvent, the amount of light absorbed increases with the concentration of the absorbing molecules (the solution's molar concentration, ccc). Beer-Lambert Equation: A = Ecl E = Molar absorption coefficient, c = Molar concentration, l = path length %T(transmittance) readings can be converted to absorbance as follows: 1 −𝐴 𝑙𝑜𝑔 𝑇 = 𝐴 𝑂𝑅 𝐴 = − 𝑙𝑜𝑔(𝑇) 𝑂𝑅 𝑇 = 10 Qualitative Photometric Assays Qualitative photometric assays help identify compounds by analyzing their absorption spectra. These methods answer the question: “What is that compound?” How It Works ➔​ Compounds absorb light at specific wavelengths, creating unique absorption patterns. ➔​ Comparing an unknown compound’s spectrum with spectra of known substances can reveal its identity. Key Features ➔​ Full-Spectrum Analysis: Provides detailed structural information. ➔​ Wavelength-Specific Analysis: Quick identification using characteristic wavelengths, like the 260/280 nm ratio for nucleic acids and proteins. Considerations ➔​ Conditions like pH and ionic strength can affect absorption. ➔​ Always document solution conditions to ensure accurate comparisons. LAB#3 ANALYSIS OF LIPIDS BY CHROMATOGRAPHY Introduction to PhosphatidylCholine (PC) Phosphatidylcholine, also known as lecithin, is a major class of phospholipids found in biological membranes, particularly abundant in egg yolk and soybeans. It consists of two fatty acid molecules attached to a glycerol backbone through ester linkages, along with a phosphorylated choline group. The basic structure of phosphatidyl choline is shown below: ​ Glycerol Backbone: Serves as the central structural unit. ​ Fatty Acid Residues: Typically, one fatty acid is unsaturated (in the β or 2 position), and the other is saturated (in the α or 1 position). ​ Choline Group: The phosphorylated choline attaches to the glycerol backbone via a phosphodiester linkage. Phosphatidylcholine is essential in biological systems for forming cell membranes, lipid bilayers, and functioning as a storage lipid in certain plants, seeds, and animals. Phosphatidylcholine Variability The fatty acid residues attached to phosphatidylcholine can vary across different species, tissues, and even diets in the case of animals. These variations offer insights into an organism's lipid metabolism and its adaptations to diet or environment. ​ Saturated Fatty Acid: Typically found in the α (1) position of the glycerol backbone. ​ Unsaturated Fatty Acid: Commonly esterified at the β (2) position. This experiment involves analyzing the fatty acids in phosphatidylcholine samples, either derived from egg yolk or soybeans, by two main techniques: 1.​ Thin Layer Chromatography (TLC) to check for the purity of the phosphatidylcholine mixture. 2.​ Gas Chromatography (GC) to analyze the fatty acid composition, following a mild alkaline hydrolysis (saponification) to release fatty acids, which are then converted to fatty acid methyl esters (FAMEs) for GC analysis. Experiment Procedures 1.​ Thin Layer Chromatography (TLC): ○​ TLC will separate the phospholipids in the mixture based on their polarity. Phosphatidylcholine, being a phospholipid, should migrate to a characteristic position when visualized. This will help determine the purity of the phosphatidylcholine fraction. 2.​ Saponification: ○​ Mild alkaline hydrolysis of phosphatidylcholine releases fatty acids from the phospholipid molecule. This step breaks the ester bonds between the fatty acids and the glycerol backbone. 3.​ Methylation:  ○​ The free fatty acids are converted into fatty acid methyl esters (FAMEs) using a methylating agent. This step is crucial because the FAMEs are volatile and can be analyzed using gas chromatography. 4.​ Gas Chromatography (GC): ○​ GC will separate the methylated fatty acids based on their molecular size and volatility. By comparing retention times of FAMEs with known standards, you can identify the different saturated and unsaturated fatty acids present in the sample. Expected Results ​ Thin Layer Chromatography will show phosphatidyl choline as a distinct spot, helping to confirm its presence and purity. ​ Gas Chromatography will reveal the composition of fatty acids, identifying the saturated and unsaturated fatty acid content in the phosphatidylcholine samples. Introduction to Chromatography Chromatography is a powerful technique used for separating components in a mixture based on their interactions with two phases: a stationary phase and a mobile phase. The principle behind chromatography is that different solutes in a mixture will interact differently with these phases, causing them to move at different rates through the stationary phase. This differential movement allows for the separation of the components of the mixture. Key Concepts in Chromatography 1.​ Phases in Chromatography: ○​ Stationary Phase: The phase that stays fixed in place and does not move. It can be a solid surface or an immobile liquid surface. ○​ Mobile Phase: The phase that moves through the stationary phase, carrying the solutes with it. It can be a liquid or a gas. 2.​ Separation Mechanism: The separation of components occurs based on their differing affinities for the stationary phase (which can be influenced by polarity, size, or charge) and the mobile phase. Some solutes will be more attracted to the stationary phase, slowing their movement, while others will be carried more readily by the mobile phase. 3.​ Rate of Migration: The rate at which a solute moves through the stationary phase is influenced by the solute's chemical nature, the composition of the solvent, temperature, and flow rate. Solutes that have a higher affinity for the mobile phase will migrate faster, while those that interact more strongly with the stationary phase will migrate more slowly. 4.​ Retention Factor (Rf): A common measure of migration in chromatography is the retention factor (Rf), which is calculated as the ratio of the distance a solute migrates to the distance traveled by the solvent:  𝑀𝑜𝑣𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑏𝑎𝑛𝑑 (𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑) 𝑅𝑓 = 𝑀𝑜𝑣𝑒𝑚𝑒𝑛𝑡 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡 (𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡) This ratio provides a way to compare the migration of different solutes. Types of Chromatography There are four basic types of chromatography, which differ based on the combination of mobile and stationary phases used: 1.​ Gas-Solid Chromatography: In this method, the stationary phase is a solid adsorbent, and the mobile phase is a gas. 2.​ Gas-Liquid Chromatography (GLC or GC): Here, the stationary phase is a liquid supported on an inert solid, and the mobile phase is a gas. This method is often used to separate volatile compounds. 3.​ Liquid-Liquid Chromatography: This method uses a liquid stationary phase and a liquid mobile phase. The separation is based on the partitioning of solutes between the two liquid phases. 4.​ Liquid-Solid Chromatography: In this method, the stationary phase is a solid, and the mobile phase is a liquid. Separation occurs based on the adsorption of solutes to the solid phase. Chromatography Formats The format of chromatography refers to how the stationary and mobile phases are set up during the separation process. Some of the common formats include: ​ Column Chromatography: The mobile phase (liquid or gas) is passed through a column filled with the stationary phase (solid or liquid). The mixture is added to the top of the column, and the solutes are separated as they move through the column. ​ Thin Layer Chromatography (TLC): In TLC, the stationary phase is a thin layer of adsorbent (usually silica gel) applied to a flat surface (glass or plastic). The mobile phase is a liquid that moves through the stationary phase by capillary action. TLC is widely used for the analysis of small amounts of substances and provides quick results. Molecular Processes in Chromatography The separation of solutes in chromatography occurs due to various molecular processes: 1.​ Partitioning: In partition chromatography, solutes are distributed between a stationary phase (often aqueous) and a moving organic phase. This is driven by differences in solubility. 2.​ Absorption: In many forms of chromatography, solutes are absorbed onto the surface of the stationary phase.  3.​ Ion Exchange: In ion exchange chromatography, solutes are separated based on their charge. The stationary phase has charged sites that attract and hold solutes of the opposite charge. 4.​ Gel Filtration: This method separates solutes based on their size. Larger molecules are excluded from the stationary phase and move faster, while smaller molecules are trapped and move slower. 5.​ Affinity Chromatography: This technique is based on the specific interaction between a solute and the stationary phase. For example, a protein may bind to an antibody immobilized on the stationary phase, allowing for selective separation. LAB#4 SEPARATION AND ANALYSIS OF BLOOD PROTEINS Introduction to Protein Roles and Structures ➔​ Proteins are highly diverse in function and structure, making up thousands of different types in a cell. ➔​ Protein abundance varies: some are present in only a few copies, while others exist in millions. ➔​ Folding: The unique folding of a polypeptide chain, determined by its amino acid sequence, gives proteins their specific functional and structural roles. Protein Structure and Function ➔​ Secondary Structure: Stabilized by hydrogen bonds between the carbonyl oxygen and the amide hydrogen within the polypeptide chain. ➔​ Tertiary Structure: Determined by: ◆​ Interchain hydrogen bonds. ◆​ Polar and nonpolar attractions. ◆​ Covalent bonds (e.g., sulfhydryl bridges). ➔​ Quaternary Structure: Found in multi-subunit proteins, further contributing to functionality. Protein function is directly linked to these intricate structures. Disruption of these levels of organization impairs or abolishes functionality. Protein Solubility ➔​ Proteins can be grouped based on solubility into categories such as: ◆​ Albumins: Water-soluble. ◆​ Globulins: Soluble in dilute salt solutions.  ◆​ Glutelins: Soluble in dilute acids or bases. ◆​ Scleroproteins (e.g., keratins, collagens): Insoluble in water. ◆​ Other categories include myosins, fibroins, fibrinogens, and elastins. ➔​ Solubility is influenced by the nature of the protein's amino acid side chains and the surrounding environment. ➔​ Four Major Factors Affecting Solubility: ionic strength, pH, temperature, and the dielectric constant of the solvent. Denaturation of Proteins ➔​ Denaturation disrupts the three-dimensional structure of a protein, rendering it nonfunctional. ➔​ Causes include: ◆​ Chemical changes: pH shifts, exposure to detergents or solvents. ◆​ Physical factors: Heat, agitation. ➔​ Denatured proteins often lose solubility as their folded structures unravel. Effects of Ionic Strength on Protein SolubilityProtein solubility in aqueous solutions depends on the interaction between protein molecules, water, and ions in the solution. These interactions are influenced by the ionic strength of the solution, which reflects the concentration and charge of dissolved ions. Key Concepts 1.​ Hydration Shell: ○​ Proteins are soluble in water due to a shell of water molecules that interact with the charged amino acid residues. ○​ This hydration shell keeps the protein molecules dispersed and soluble in solution. 2.​ Ionic Strength Formula:​ 1 2 𝑢 = 2 𝐶𝑖𝑍𝑖 ○​ Ci: Molar concentration of ion iii. ○​ Zi: Number of charges on ion iii. ○​ Higher charges (Zi​) and concentrations (Ci) increase ionic strength. 3.​ Ion Atmosphere: ○​ Ions in solution create an "ion atmosphere" that affects protein solubility. ○​ Divalent and trivalent ions have a stronger influence than monovalent ions. Salting-In and Salting-Out Effects  1.​ Salting-In: ○​ At low ionic strength, added neutral salts (e.g., ammonium sulfate) stabilize protein molecules by interacting with their charged groups. ○​ This prevents aggregation and increases protein solubility. 2.​ Salting-Out: ○​ At high ionic strength, excessive salt competes with proteins for water molecules. ○​ This reduces the hydration shell around the proteins and neutralizes their charges. ○​ Proteins become less polar and aggregate, leading to precipitation. Demonstration with Blood Plasma Proteins Using ammonium sulfate, proteins are sequentially precipitated based on their solubility: 1.​ Fibrinogens: Precipitate at low ammonium sulfate concentrations (least soluble). - Clotting proteins. 2.​ Globulins: Precipitate as more salt is added.- Proteins for metabolism, transport, and immunity. 3.​ Albumins: Precipitate at saturation levels of ammonium sulfate (most soluble). - Proteins regulating osmotic pressure and transport Normal blood plasma contains 6.5–7.5 g of protein per 100 mL. Albumins, often constituting over 50% of blood plasma proteins, are isolated in the final step. Salting-Out with Ammonium Sulfate Ammonium sulfate (AS) is highly soluble and widely used for protein precipitation due to its ability to alter protein solubility at varying concentrations. Fractionation Basis ​ Proteins are fractionated based on their differential solubility at specific ammonium sulfate saturations: ○​ Fibrinogen precipitates at low AS saturation. ○​ Globulins require moderate AS saturation to precipitate. ○​ Albumins precipitate only at high AS saturation. 50.6[𝑆2−𝑆1] 𝑉1[𝑆2−𝑆1] A)​𝑋 = 1−0.3𝑆2 B) 𝑌 = 1−𝑆2 X = grams of solid AS to be added to 100 mL of solution of S1 saturation to change it to S2 saturation (at 0oC) Y = mL of saturated AS required to be added to V1 (at 0oC) Separation of Proteins by Polyacrylamide Gel Electrophoresis (PAGE) Purpose This lab introduces polyacrylamide gel electrophoresis (PAGE) as a technique to separate proteins based on molecular mass. Proteins from fractions of blood serum precipitated by ammonium sulfate (from the previous experiment) are analyzed to observe the presence of distinct protein types. Principle of PAGE 1.​ Gel Composition: ○​ Proteins migrate through a polyacrylamide gel matrix. ○​ The gel acts as a sieve, with smaller proteins migrating faster than larger ones. 2.​ Charge-Based Separation: ○​ Proteins carry a charge in the presence of an electrical field. ○​ Migration occurs toward the anode (+ pole) due to the induced negative charge on proteins. 3.​ Molecular Weight: ○​ Migration rate is inversely proportional to molecular weight. Smaller proteins travel farther. SDS-PAGE: Role of Sodium Dodecyl Sulfate (SDS) ​ SDS: ○​ A negatively charged detergent that binds to proteins, disrupting secondary, tertiary, and quaternary structures. ○​ Denatures proteins into linear polypeptide chains, masking intrinsic charges and conferring a uniform negative charge proportional to the length of the polypeptide. ​ β-Mercaptoethanol: ○​ A reducing agent that breaks disulfide bridges in proteins. ○​ Ensures complete denaturation and separation of protein subunits. Experimental Procedure 1.​ Sample Preparation: ○​ Protein fractions are mixed with: ​ SDS: Denatures proteins. ​ β-Mercaptoethanol: Reduces disulfide bonds. ○​ Results in negatively charged, linear protein chains. 2.​ Electrophoresis Setup: ○​ Proteins are loaded onto the gel. ○​ An electrical current is applied. ○​ Proteins migrate toward the anode due to their negative charge.  3.​ Separation: ○​ Proteins separate based on molecular size: ​ Smaller proteins migrate faster and farther. ​ Larger proteins migrate more slowly. 4.​ Visualization: ○​ After electrophoresis, the gel is stained to visualize protein bands. ○​ Staining highlights the distinct bands corresponding to proteins of different molecular weights. Applications ​ PAGE is widely used in: ○​ Protein purification: Assessing the purity of samples. ○​ Molecular biology: Identifying proteins of interest. ○​ Clinical diagnostics: Investigating protein anomalies in blood or other tissues. The Lowry Assay for Protein Estimation The Lowry method is a two-step assay used to determine the protein concentration in a solution with greater sensitivity than the biuret method. Its basis lies in two key reactions: 1.​ Biuret Reaction: ○​ Proteins react with copper ions (Cu²⁺) in an alkaline solution to form a blue-violet complex. ○​ This reaction involves peptide bonds in the protein. 2.​ Reduction of the Folin-Ciocalteu Reagent: ○​ The Folin reagent is reduced by specific amino acids in the protein, particularly tyrosine and tryptophan residues. ○​ This produces an intense blue color, significantly increasing assay sensitivity. Advantages 1.​ Increased Sensitivity: ○​ The Lowry method can detect protein concentrations as low as 1 µg/mL, compared to the biuret method's minimum sensitivity of 0.25 mg/mL. 2.​ Widespread Use: ○​ Despite its limitations, the Lowry method is widely applied because it combines high sensitivity with relatively straightforward execution. Disadvantages 1.​ Variability with Different Proteins:  ○​ Color development depends on the tyrosine and tryptophan content, which varies among proteins. This leads to less consistent results compared to the biuret method. 2.​ Nonlinear Response: ○​ The color development is linear only within a narrow range of protein concentrations, making accurate quantification challenging outside this range. 3.​ Interference by Other Substances: ○​ Certain biological molecules can interfere with the reaction, either by: ​ Producing false positives: Substances with phenolic groups (e.g., phenols, flavonoids) can reduce the Folin reagent and inflate protein estimates. ​ Inhibiting the reaction: High concentrations of ammonium sulfate (>0.15%) inhibit color development. LAB #5 ENZYME KINETICS + INHIBITION Enzyme Function and Regulation Overview Enzymes are critical in facilitating chemical reactions in living cells, with each type catalyzing specific reactions. They largely determine the cell's activity and structure. Regulation of Enzyme Activity ➔​ Transcriptional and Translational Control: ◆​ Only a subset of genes for enzymes are transcribed and translated, limiting the number of enzyme molecules in a cell. ➔​ Post-Translational Control: ◆​ Inhibitors: Molecules that reduce enzyme activity by binding to the active site (competitive) or other sites (non-competitive). ◆​ Activators: Molecules that increase enzyme activity. ◆​ Covalent Modification: Enzymes are turned on or off by chemical changes (e.g., phosphorylation) catalyzed by other enzymes. Acid Phosphatase ➔​ Model System:​ Acid phosphatase from wheat germ is used to study: ◆​ Enzyme kinetics. ◆​ Effects of competitive and non-competitive inhibition. ◆​ Influence of pH on enzyme activity, demonstrating how tertiary protein structure impacts function. ➔​ Biological Significance: ◆​ Found in plants, animals, and bacteria.  ◆​ In humans: ​ Produced in tissues like the liver, bone marrow, and spleen. ​ High levels in the prostate gland; used as a marker for prostate cancer or forensic detection of seminal fluid. Effect of Substrate Concentration on Enzyme Reaction Rate Enzyme Saturation: 1.​ Enzymes bind to substrates to catalyze reactions. 2.​ At low substrate concentrations, reaction rates increase as substrate concentration increases. 3.​ At high substrate concentrations, enzymes become saturated, and the reaction rate plateaus. Competitive Inhibition of Acid Phosphatase Activity Key Concepts ➔​ Competitive Inhibition: ◆​ Molecules with a shape similar to the substrate can bind to the enzyme's active site. ◆​ These molecules compete with the substrate, temporarily preventing the enzyme from catalyzing the reaction. ◆​ The inhibition is reversible and depends on substrate and inhibitor concentrations. ➔​ Role of KH₂PO₄: ◆​ KH₂PO₄ provides free phosphate ions that compete with the substrate (p-nitrophenyl phosphate, pNPP) for the enzyme's active site. ◆​ Enzyme activity decreases as KH₂PO₄ occupies the active sites. Expected Results: ➔​ At low substrate concentrations, competitive inhibition is strong because most enzyme active sites are occupied by the inhibitor. ➔​ As substrate concentration increases, substrate molecules outcompete the inhibitor, and enzyme activity increases. LAB #6 VISUALIZATION OF CELLS USING LIGHT MICROSCOPY AND HISTOLOGICAL STAINING TECHNIQUES Introduction to Histology Histology is the study of the structure of cells and tissues in relation to their specialized functions. The main technique in histology is the preparation of tissue sections for microscopic examination. The process includes the following steps: 1. Fixation: ​ Purpose: To preserve tissue structure and prevent decomposition after death. ​ Common Fixatives: Formalin and glutaraldehyde. ​ Goal: Fixation should be done quickly to minimize post-mortem changes in the cells. 2. Dehydration: ​ Purpose: To remove all traces of water, as embedding materials (like paraffin) are not miscible with water. ​ Process: Tissues are passed through a series of increasing concentrations of alcohol (50%, 60%, 70%, 80%, 90%, and 100% ethanol). ​ Goal: Gradual dehydration to avoid distortion of tissues. 3. Embedding: ​ Purpose: To provide support for thin sectioning of the tissue. ​ Process: Tissues are first cleared using a solvent (e.g., xylene) that is miscible with both alcohol and paraffin. The tissue is then infiltrated with molten paraffin and embedded in molds. ​ Goal: To create hardened tissue blocks that are easy to cut. 4. Sectioning: ​ Purpose: To cut tissue into thin sections (5-10 μm) for microscopic examination. ​ Process: Tissues are cut using a microtome, then mounted onto microscope slides. ​ Goal: To create thin, precise sections for observation. 5. Staining: ​ Purpose: To provide contrast for better visualization of tissue structures, as most cells are transparent without staining. ​ Process: Two contrasting stains (a primary stain and a counterstain) are often used together. ​ Types: ○​ In vitro staining: Staining of fixed and embedded tissues. ○​ In vivo (vital) staining: Staining of living tissues to reveal details that are not visible in fixed tissues. Components of H&E Staining:  1.​ Hematoxylin: ○​ Source: A natural dye extracted from the heartwood of the logwood tree (Haematoxylon). ○​ Staining Effect: Hematoxylin stains cell nuclei a purple to dark blue color. 2.​ Eosin: ○​ Source: A chemical derivative of the fluorescent compound fluorescein. ○​ Staining Effect: Eosin stains the cytoplasm, connective tissue, muscle, and extracellular substances such as collagen, giving them a pink or red color. Plant Tissue Types Plants have three main types of tissue that make up their modular body: 1.​ Dermal Tissue: ○​ Composition: Includes the epidermal layers and trichomes (hairs) on the surface of the plant. ○​ Function: Often excretes hydrophobic waxes like cutting to waterproof the plant, protecting it from water loss and environmental factors. 2.​ Vascular Tissue: ○​ Composition: Made up of specialized, elongated cells that are essential for transporting water and nutrients. ○​ Function: Transports water and nutrients between the roots and leaves. It consists of xylem (water transport) and phloem (nutrient transport). 3.​ Ground Tissue: ○​ Composition: Includes cells that can store nutrients, provide structural support, and perform photosynthesis. ○​ Function: Plays roles in storage, support, photosynthesis, and repair. These cells can also dedifferentiate and redifferentiate into other cell types when needed. Vascular Tissue Organization ➔​ Vascular Stele: ◆​ Vascular tissue is organized into steles, which are cylindrical masses of cells that contain both xylem and phloem. ◆​ In flowering plants, the vascular stele is divided into vascular bundles that permeate through the stem and lateral organs (including leaves). ◆​ Vascular Bundles: These are the individual strands of the vascular tissue, and within each bundle, you will see both xylem and phloem. LAB #7 CELLULAR RESPIRATION Introduction: Cellular Respiration in Saccharomyces cerevisiae ➔​ Two Main Energy Strategies: ◆​ Photosynthesis: Used by plants to produce energy from sunlight. ◆​ Cellular Respiration: Used by chemotrophic (heterotrophic) organisms to produce energy by breaking down organic molecules. ➔​ Aerobic Cellular Respiration: ◆​ Occurs in organisms that require oxygen. ◆​ Organic molecules (e.g., glucose) are completely oxidized into carbon dioxide (CO2) and water (H2O). ◆​ Energy (ATP) is generated in the process. ◆​ Chemical equation:​ C6H12O6 + 6O2 → 6CO2 + 6H2O + energy (ATP) ➔​ Anaerobic Cellular Respiration: ◆​ Occurs in organisms that do not need oxygen. ◆​ Glucose is broken down into ethanol (C2H5OH) and carbon dioxide (CO2), producing small amounts of ATP. ◆​ Chemical equation:​ C6H12O6 → 2CO2 + 2C2H5OH + energy (ATP) ➔​ Saccharomyces cerevisiae (Brewer’s Yeast): ◆​ Facultative anaerobes: Yeast can survive and grow with or without oxygen. ◆​ Under aerobic conditions, they perform aerobic respiration. ◆​ Under anaerobic conditions, they perform fermentation, producing ethanol and CO2. Part 1: Comparing Ethanol Production by Yeast in Aerobic and Anaerobic Conditions Method 1: Specific Gravity Measurements ➔​ Specific gravity is a measure of the density of a solution compared to water. ➔​ Water has a specific gravity of 1.000, and pure ethanol has a specific gravity of 0.785. ➔​ Why Use Specific Gravity? As fermentation occurs, glucose is converted to ethanol, which reduces the density of the solution. This causes a decrease in the specific gravity. 1.​ Steps: a.​ Initial Measurement: i.​ Use a hydrometer to measure the specific gravity of the yeast culture before fermentation (e.g., aerobic culture). ii.​ Fill a hydrometer cylinder halfway with the solution, gently place the hydrometer in the liquid, and read the value at the top of the meniscus. Repeat for anaerobic culture.  b.​ Final Measurement: i.​ After fermentation, measure the specific gravity again. c.​ Calculate Ethanol Content: i.​ Percent Alcohol by Weight = (Original S.G. − Final S.G.) × 105 Method 2: Alcohol Dehydrogenase (ADH) Reaction ➔​ Alcohol Dehydrogenase (ADH) enzyme oxidizes ethanol, converting NAD+ to NADH. ➔​ NADH absorbs light differently than NAD+, so we can measure the ethanol content by detecting absorbance changes using a spectrophotometer. ➔​ RCH2OH (ethanol)+NAD+→RCHO (aldehyde) -​ The amount of NADH produced is directly related to the amount of ethanol in the sample. Method 3: Gas Chromatography for Ethanol Content Gas chromatography (GC) separates and measures different compounds, including ethanol. Steps: 1.​ Samples: The lab technician has already run your aerobic and anaerobic yeast samples through the GC. 2.​ Standard Curve: You will be given a standard curve with known ethanol concentrations. 3.​ Determine Ethanol: Compare the peaks in your sample chromatograms to the standard curve to calculate the ethanol content in both cultures. Calculating Glucose Content by Glucose Oxidase Colorimetric Reaction This method uses glucose oxidase to break down glucose and produce a coloured product that can be measured with a spectrophotometer. Steps in the Reaction: ➔​ Glucose Breakdown: Glucose oxidase reacts with glucose and oxygen to form gluconolactone and hydrogen peroxide (H2O2). -​ Glucose + O2 → Gluconolactone + H2O2 ➔​ Colour Production: The enzyme peroxidase reacts with O-Dianisidine and hydrogen peroxide to form a coloured product. -​ O-Dianisidine + H2O2 → Oxidized O-Dianisidine (coloured product) ➔​ The intensity of the colour produced is measured using a spectrophotometer. The more intense the colour, the higher the glucose concentration. LAB #8 RESPIRATION IN ISOLATED MITOCHONDRIA Keilin-Hartree Mitochondrial "Particles" ➔​ Keilin-Hartree Particles are fragments of the inner mitochondrial membrane that can perform electron transport, but they cannot produce ATP (phosphorylate ADP) by themselves. ➔​ Why No ATP Production? ◆​ These particles can transfer electrons but lack the full machinery needed for ATP production. Specifically, they don't have the proper proton gradient or ATP synthase working as it would in intact mitochondria. Key Points of the Electron Transport Chain: 1.​ Electron Entry: a.​ NADH donates electrons to Complex I. b.​ FADH2 donates electrons to Complex II. c.​ Electrons from both Complex I and Complex II are passed to Ubiquinone (Coenzyme Q). 2.​ Electron Flow: a.​ Ubiquinone passes electrons to Complex III. b.​ Complex III transfers electrons to Cytochrome c. c.​ Cytochrome c passes electrons to Complex IV. d.​ Complex IV transfers electrons to oxygen, forming water. 3.​ ATP Production: a.​ As electrons move through the chain, protons (H+) are pumped across the membrane, creating a proton gradient. b.​ ATP Synthase uses this gradient to produce ATP. Inhibition Points: ➔​ Complex I can be inhibited by rotenone (blocks NADH entry). ➔​ Complex II can be inhibited by malonate (blocks FADH2 entry). ➔​ Complex III can be inhibited by antimycin A (blocks electron transfer from ubiquinone). ➔​ Complex IV can be inhibited by cyanide or carbon monoxide (blocks electron transfer to oxygen). Succinate enters the electron transport chain via Complex II (distinct from other intermediates that use Complex I). Malonate inhibits Complex II (blocking succinate oxidation), while Antimycin A inhibits Complex III (blocking electron flow to oxygen). These inhibitors disrupt electron transfer, blocking the production of ATP through oxidative phosphorylation. Oxygen Electrodes ➔​ Purpose: Measure how much oxygen mitochondria are using, which shows how active the electron transport is. ➔​ How it Works: ◆​ The oxygen electrode has two parts: an anode and a cathode in a chamber filled with liquid. ◆​ The electrode is placed in a sample chamber with a liquid containing oxygen (like a mitochondrial solution). ➔​ Oxygen Diffusion: ◆​ Oxygen from the sample chamber moves through a membrane into the electrode chamber. ◆​ The rate of oxygen movement depends on how much oxygen is in the sample chamber. ➔​ Current and Oxygen Use: ◆​ The cathode reduces oxygen to water, and this creates a current. ◆​ If the mitochondria use oxygen, the oxygen concentration drops, and the current changes. ◆​ The system measures this change in current, which tells us how much oxygen the mitochondria are using. If oxygen levels drop, it means mitochondria are using it for energy, and the electrode records this.