4002 (4 Practical)

• Bradford Protein Assay is a biochemical technique used to measure the total protein concentration in a sample.
• The enzyme inase serves as the test sample in assays.
• Coomassie Brilliant Blue dye binds to proteins, causing a color change from brown to blue.
• This dye primarily interacts with arginine, lysine, and histidine residues
• The intensity of the blue color directly correlates with protein concentration.
• Protein concentration is gauged at a wavelength of 595 nm using spectrophotometry.

Protein Standards Prep

• Protein standards of known concentrations (0-600 µg/mL) are compared against unknown protein samples.
• Label six small test tubes for protein standards, each with a total volume of 1 mL.
• Vary the amounts of protein standard solution (0 to 1 mL).
• Use Sodium Chloride solution as a diluent to adjust the total volume to 1 mL in each tube.

Transferring Protein Standards

• Transfer 100 µL of each protein standard in duplicate into larger test tubes.
• The blank (0 µg/mL) has three replicates, while all other protein concentrations have two replicates.

Adding Bradford Reagent

• Use a glass pipette to measure 5 mL of Bradford reagent and add it to each test tube.
• Gently mix the solutions and observe the color change.
• Tubes with higher protein concentrations should appear darker blue.

Measuring Absorbance

• Use a spectrophotometer to measure absorbance at 595 nm.
• Turn on the spectrophotometer and set the wavelength to 595 nm.
• Zero the spectrophotometer using the blank sample in a cuvette, with the clear side facing the detector.
• Rinse the cuvette between samples to prevent contamination.

Preparing Inase Dilutions

• Label three small ignition tubes for different inase dilutions: 1:2, 1:5, and 1:10.
• Calculate the correct volumes of enzyme solution and diluent for each dilution, bringing each tube to 1 mL total volume.

Transferring Enzyme Dilutions

• Prepare six large test tubes and transfer 100 µL of each dilution into the larger tubes using an automatic pipette.
• Change the pipette tip between dilutions to prevent contamination.

Adding Bradford Reagent to Enzyme Dilutions

• Add 5 mL of Bradford reagent to each of the six test tubes containing inase dilutions.
• Gently mix and observe the color changes.

Measuring Enzyme Dilution Absorbance

• Measure the absorbance of each dilution in the spectrophotometer, starting with the most diluted sample (1:10).
• Record the absorbance values for each dilution in your practical booklet.

Workspace Clean Up

• Turn off the spectrophotometer.
• Clean the spectrophotometer and surrounding area using wipes.
• Clean pipettes and tip boxes.
• Place all used test tubes into a single rack for easy disposal.

Enzyme Kinetics Practical on Invertase Activity

• Enzymes are biological catalysts that speed up chemical reactions.
• Invertase catalyzes the hydrolysis of sucrose into glucose and fructose.
• Temperature, pH, and substrate concentration affect enzyme activity.
• Low temperatures slow enzyme activity, while increasing temperature raises activity to an optimum.
• High temperatures denature enzymes, reducing their effectiveness.
• Dinitrosalicylic acid (DNS) stops the reaction by reacting with glucose and fructose.

Preparing the Tubes

• Label 14 tubes for 7 temperatures (0°C, Room Temperature, 30°C, 40°C, 50°C, 70°C, 100°C), with two tubes per temperature.

Adding the Enzyme

• Add 0.5 mL of invertase enzyme solution to each tube, starting with the 0°C tubes.

Equilibrating the Tubes

• Place the 0°C tubes in an ice bath, wait 5 minutes, and then add 0.5 mL of sucrose to start the reaction.

Stopping the Reaction

• After 5 minutes, stop the reaction by adding 1 mL of DNS, shaking the tubes gently.

Multiple Temperatures

• Use a time chart to manage additions.
• Start timer when placing the tubes in the water bath
• After 5 minutes, add 0.5 mL of sucrose to start the reaction.
• At 10 minutes, stop the reaction by adding 1 mL of DNS.

Experiment Procedure

• Start the timer when placing the 30°C tubes in the water bath.
• At 3 minutes, place 40°C tubes in their water bath.
• At 5 minutes, add sucrose to the 30°C tubes.
• At 8 minutes, add sucrose to the 40°C tubes.
• At 10 minutes, stop the 30°C tubes’ reaction by adding DNS.

Final Steps

• Shake all tubes after adding DNS.
• Dispose of materials properly and clean the work space.

Functional Group Identification

• Identifying functional groups helps classify substances.
• Qualitative chemical testing involves reactions that produce color shifts or precipitates.
• Ferric Chloride Test detects phenols via color changes.

Ferric Chloride

• Ferric chloride (FeCl₃) dissolves in water to yield Fe³⁺ ions. Coordination complex leads to color change.

Test Procedure

• Prepare two test tubes with phenol and 2-Naphthol.
• Add 1 mL of deionized water and stir to dissolve.
• Add a few drops of 2.5% aqueous ferric chloride solution to each tube.
• Observe for color changes: red, blue, purple, or green indicates the presence of a phenol.
• Further testing may be required for verification.

Acid-Base Titration

• Titration determines the concentration of an unknown acid or base by reacting it with a solution of known concentration.
• A titrant of known concentration is added to the analyte until the reaction reaches completion.
• The equivalence point, where moles of acid and base are equal, is detected with indicators or pH meters.
• The concentration of an unknown acid or base is determined using the titration formula: M1V1=M2V2.
• Pharmaceuticals, food industry, environmental science, and biochemistry all utilize acid-base titrations.

Preparation

• Gather Potassium hydrogen phthalate (KHP), Sodium hydroxide (NaOH), Phenolphthalein indicator etc.
• Clean the burette with deionized water.

Weighing and Dissolving KHP

• KHP will influence the molarity calculations.
• Transfer the measured KHP to a flask containing approximately 100 mL of deionized water.

Setting Up the Burette

• Rinse the burette with the NaOH solution to remove water.
• Fill the burette with NaOH solution and record the initial volume.
• Remove any air bubbles from the burette tip.

Titration Process

• Add a few drops of phenolphthalein to the KHP solution as an indicator.
• Slowly add NaOH from the burette while swirling the flask.
• The endpoint is reached when the solution remains a faint pink color for at least 30 seconds.

Calculations

• Determine the moles of KHP used.
• Using the volume of NaOH, calculate the molarity of the NaOH solution.

Best Practices

• Estimate the approximate volume of NaOH required before doing precise titrations.
• Use a white background under the flask to observe the color change.
• Repeat the titration at least three times.
• Clean equipment to avoid contamination.

Titration Results

• The indicator shows a color change at the equivalence point of the titration.
• Phenolphthalein turns from colorless to pink when the pH is above 7.
• You calculate the concentration of the unknown solution using the formula C1V1=C2V2.

Functional Groups - Results

• Reactions with reagents indicate functional groups, causing color changes, precipitates, or gas formation.
• Aldehydes and ketones can be identified using Tollens' reagent (silver mirror formation) or Fehling’s solution (red precipitate).
• Alcohols react with Lucas reagent to form an emulsion.
• Carboxylic acids react with sodium bicarbonate to release carbon dioxide gas.
• Alcohols show an IR absorption peak around 3300 cm⁻¹ for the O-H stretch.
• Carbonyl groups show a sharp peak around 1725 cm⁻¹.

Enzyme Kinetics - Results

• Enzyme activity increases with temperature, up to an optimal point, then declines due to denaturation.
• The optimal temperature has a steady increase in activity.
• After this point, the enzyme denatures, and the rate of reaction drops sharply.
• In Arrhenius equation k=Ae−EaR, k is the rate constant, A is the pre-exponential factor, Ea is the activation energy,R is the gas constant, and T is temperature in Kelvin.

Bradford Assay - Results

• Protein solution is mixed with Coomassie Brilliant Blue dye, resulting in a color change from brown to blue.
• The intensity of the blue color is directly proportional to the protein concentration.
• Calculate the protein concentration in unknown sample by finding its corresponding value from the standard curve: Concentration=Absorbance−InterceptSlope.

Formulas

• C where subscript 1V subscript 1 equals C subscript 2 V Subscript 2 : C1V1=C2V2 is used to calculate concentration.
• k equals A e to the power of negative E subscript a over RT : k=Ae−EaRT details Arrhenius equation.
• Concentration equals Absorbance minus Intercept over Slope: Concentration=Absorbance−InterceptSlope​ is used to calculate protein levels.

Questions and Answers

• Potassium hydrogen phthalate (KHP) is an organic salt.
• Phenolphthalein is colorless in acidic solutions and pink in alkaline solutions.
• Molecular weight of aspirin (C₉H₈O₄) is 180 g/mol.
• In titration, Attempt 1 and Attempt 2 are within the 1% limit.
• A tablet weighing 0.5065 g, titrated with 17.50 cm³ of 0.1002 M NaOH solution has 62.5% of aspirin.
• Proteins give rise to the precipitation observed on adding acetic acid to milk.
• The Tollen’s test identifies carbohydrates.
• Two compounds show a purple color change with ferric chloride.
• Three compounds dissolve in dilute sodium hydroxide.
• Compound B possesses a peptide bond but NOT a phenolic functional group.
• Determining protein concentration of a sample involves: reading its A595nm and multiplying protein concentration by 2.
• Non-specific absorbance is mainly due to the absorbance of uncomplexed dye.
• In Bradford assay, the chromophore is affected by the proportion of basic amino acids in the protein.
• The UV absorption method does not require denaturing the sample.
• Tyrosine and tryptophan reacts with the Folin-Ciocalteau reagent in protein work.
• Invertase is an enzyme produced by microorganisms.
• On a glucose molecule C1 is the anomeric carbon.
• DNS measures sugar concentration, NaOH alters pH, and temperature dependency graph peaks related to the balance between temperature and enzyme activity
• At 19°C is 0.06 A540nm/min. The reaction between invertase and sucrose was stopped by Sodium Hydroxide.
• 2mg/ml is equivalent to 1200 mg/L.
• In the Bradford assay for protein, the chromophore is affected by the proportion of basic amino acids in the protein and sample is not denatured.
• Three compounds contain a peptide bond possess sufficiently acidic functional groups like phenols or carboxylic acids.
• 1 ml of analyte at a 1 in 10 dilution can be created with 0.1 ml analyte + 0.9 ml diluent.
• If a sample was weighed to 4 decimal places (e.g., 0.0785g), there are 3 significant figures.
• Primary standards should not be hygroscopic as that alters its mass and concentration.