P2 POST LAB BIOCHEMISTRY (1).pptx

BIOCHEMISTRY P2 POST LABORATORY Pre p a re d b y: E n n a M a r i e G i c a , R P h  ISOLATION AND CHARACTERIZATION OF PROTEINS Objectives: 1.To i s o l a t e p r o t e i n s 2.To o b s e r v e t h e i r p h y s i c a l properties and compare with theoretical results 3.To c o n d u c t t e s...

BIOCHEMISTRY P2 POST LABORATORY Pre p a re d b y: E n n a M a r i e G i c a , R P h  ISOLATION AND CHARACTERIZATION OF PROTEINS Objectives: 1.To i s o l a t e p r o t e i n s 2.To o b s e r v e t h e i r p h y s i c a l properties and compare with theoretical results 3.To c o n d u c t t e s t s t h a t i d e n t i f y s p e c i fi c p r o t e i n s b a s e d o n c o l o r reactions Experiments and Observations Isolation of Gluten from Wheat Objective: Extract gluten, a protein, from wheat flour Materials: 100g Wheat flour, Iodine solution, Theoretical Result: Cheesecloth Gluten is the insoluble protein left after starch is removed Procedure: Observation: Sticky, elastic gluten remains after 1. Mix flour with water to make dough washing 2. Knead and wrap in cheesecloth, wash under Color Change: cold water to remove starch Iodine changes to blue/black in the presence of 3. Test the washings with iodine solution starch 4. Dry and weigh the remaining gluten After washing, the color should not change when iodine is added, indicating starch removal. Rationale: Iodine reacts with starch to form a blue/black complex. As starch is removed during washing, gluten (which does not react with iodine) remains, so the iodine no longer changes color. Isolation of Bean Protein Color Change: Objective: Extract proteins from beans The precipitated protein does not react with iodine, indicating it is free of starch Materials: 1 cup soaked beans, Acetic acid, Rationale: Cheesecloth Proteins precipitate when their solubility is Procedure: reduced by acid. Acetic acid disrupts the 1. Grind soaked beans with water protein’s structure, causing it to clump 2. Filter through cheesecloth to separate solids together and form a visible precipitate 3. Add acetic acid to precipitate the protein 4. Dry and weigh the precipitate Theoretical Result: Proteins like globulins are precipitated by acid Observation: A white or off-white solid forms after adding acid  Isolation of Albumin from Egg White Isolation of Myoglobin from Muscle Objective: Isolate albumin from egg white Objective: Isolate myoglobin, a muscle protein Materials: Egg white, Acetic acid, NaCl Materials: Minced beef, Ammonium sulfate Procedure: solution 1. Separate the egg white and weigh it Procedure: 2. Add acetic acid and NaCl to precipitate the 1. Mix minced beef with ammonium sulfate albumin 2. Filter through cheesecloth to obtain a red 3. Filter, dry, and weigh the protein myoglobin solution Theoretical Result: 3. Centrifuge the extract for purification Albumin precipitates out as a white solid Theoretical Result: Myoglobin is the red, oxygen-carrying Observation: White, gel-like substance forms, protein in muscle which is the albumin Observation: A dark red liquid appears, Color Change: containing myoglobin No specific color change in the basic Color Change: No significant color change as myoglobin is isolation process, but the albumin solidifies and becomes visible naturally red Rationale: Rationale: Salt (NaCl) helps stabilize protein Myoglobin binds oxygen, giving muscle its precipitates, while acetic acid causes the red color. It remains soluble in the albumin to clump together, forming the ammonium sulfate solution, allowing for its visible white precipitate extraction and purification Qualitative Tests for Protein Identifi cation Biuret Test (General Test for Protein) Ninhydrin Test (Test for Free Amino Groups) Principle: The Biuret test detects peptide bonds in proteins Principle: Detects free amino groups in Procedure: Add sodium hydroxide amino acids and proteins (NaOH) and copper sulfate (CuSO4) to Procedure: Add ninhydrin solution to the the protein sample protein sample, heat gently Theoretical Result: Theoretical Result: Color Change: A purple/violet Color Change: A blue/purple color color appears, indicating the develops if free amino groups are presence of peptide bonds present. Exception: Proline gives a yellow Rationale: Copper ions (Cu²⁺) react with the color due to its unique structure peptide bonds in proteins, forming Rationale: Ninhydrin reacts with amino groups to a purple complex. The intensity of the color reflects the concentration form a colored complex. It’s a sensitive of proteins test for detecting amino acids and peptides Xanthoproteic Test (Test for Aromatic Amino Acids) Millon’s Test (Specific Test for Tyrosine) Principle: Detects proteins containing aromatic amino acids like tyrosine and Principle: Detects phenolic groups, tryptophan particularly tyrosine Procedure: Add concentrated nitric acid Procedure: Add Millon’s reagent and (HNO₃), then neutralize with sodium heat hydroxide (NaOH) Theoretical Result: Theoretical Result: Color Change: A red color Color Change: A yellow/orange indicates the presence of color indicates the presence of tyrosine aromatic amino acids Rationale: Rationale: Millon’s reagent reacts with the Nitric acid reacts with the aromatic phenolic group of tyrosine, rings of tyrosine and tryptophan, producing a red-colored forming a yellow nitro derivative. complex The yellow color deepens to orange upon neutralization  Post-Laboratory Analysis of Results Rationale Summary: Color reactions occur due to the interaction of reagents with specific groups in proteins Purple in Biuret test: Indicates peptide bonds, a hallmark of protein structure Blue/Purple in Ninhydrin: Indicates free amino groups in amino acids Yellow in Xanthoproteic: Shows the presence of aromatic amino acids like tyrosine Red in Millon’s test: Specific for the phenolic group in tyrosine  Enzyme Experiment Extraction of Salivary Amylase Objective: Extract amylase enzyme from saliva and demonstrate its ability to break down starch Materials: Collected saliva Starch solution Iodine solution Buffer solutions Procedure: 1.Collect saliva by rinsing with water and spitting into a beaker 2.Mix saliva (containing amylase) with starch solution 3.Test for starch breakdown by adding iodine to the mixture every 2 minutes Theoretical Result: Amylase breaks down starch into smaller sugar molecules (maltose/glucose) Observation: As starch is broken down, iodine will not form the characteristic blue/black complex, indicating starch is being digested Eventually, the blue/black color disappears as all starch is converted to sugar Rationale: Amylase hydrolyzes starch into smaller polysaccharides and glucose. Iodine reacts with starch, producing a blue/black color. When starch is broken down, iodine no longer reacts, indicating the enzyme activity Influence of pH on Amylase Activity Objective: Determine the effect of pH on the activity of salivary amylase by measuring how quickly it breaks down starch at different pH levels Materials: Salivary amylase solution (collected earlier) Starch solution Buffer solutions of varying pH Iodine solution Water bath (maintained at 37°C) Procedure: 1.Prepare 8 test tubes with buffers at different pH levels 2.Add starch and salivary amylase to each tube 3.Every 2 minutes, take a drop from each tube and mix with iodine solution on a spot plate 4.Record the time when the blue/black color (indicating starch presence) disappears Theoretical Result: Optimum pH for amylase activity is around 7.0 (neutral pH) Observation: At pH values close to 7.0, the starch breaks down fastest (color disappears quickly) At very low or high pH values, starch digestion is slower or doesn't occur at all (blue/black color persists longer) Color Change: Iodine reacts with starch to produce a blue/black color. When starch is broken down by amylase, this color will disappear Rationale: Enzymes have an optimum pH range where they function best. For amylase, neutral pH (~7) is optimal because it is similar to the pH in the human mouth. At extreme pH levels, the enzyme denatures (loses its shape), reducing its ability to break down starch Interpretation: Optimum pH: pH 7.0 is where the enzyme works the fastest (6 minutes for starch breakdown) pH extremes: Enzyme activity decreases as you move away from pH 7.0, indicating that the enzyme is less effective at acidic (pH 5.3) or basic (pH 8.0) conditions Action of Liver Catalase on Hydrogen Peroxide Objective: Demonstrate the action of catalase (an enzyme found in the liver) by breaking down hydrogen peroxide into water and oxygen Materials: Chicken liver Hydrogen peroxide (H₂O₂) Procedure: 1.Prepare a liver extract by blending chicken liver with water 2.Add hydrogen peroxide to the liver extract 3.Observe the reaction, which releases oxygen (bubbles) Theoretical Result: Catalase breaks down hydrogen peroxide into water and oxygen Observation: Immediate bubbling as oxygen gas is released from the reaction Rationale: Catalase is an enzyme present in many cells, including liver cells. It speeds up the breakdown of hydrogen peroxide, which is harmful to cells, into harmless water and oxygen. The bubbling is due to the release of oxygen gas Summary of Color Changes and Observations in Enzyme Experiments 1.S a l i v a r y A m y l a s e ( S t a r c h B r e a k d o w n ) : 1.B l u e / b l a c k c o l o r d i s a p p e a r s a s s t a rc h i s b ro ke n d o w n 2.Fa s t e s t b re a k d o w n a t n e u t r a l p H ( 7. 0 ) 2.L i v e r C a t a l a s e : 1.B u b b l i n g i n d i c a t e s b re a k d o w n o f h y d ro g e n p e rox i d e i n t o ox y g e n a n d w a t e r Post-Laboratory Questions and Rationales 1.W h y i s t e m p e r a t u r e m a i n t a i n e d a t 3 7 ° C ? 37°C is close to the normal body temperature, where e n z y m e s l i ke a m y l a s e a n d c a t a l a s e f u n c t i o n o p t i m a l l y. H i g h e r or lower temperatures may slow down or denature the enzymes. 2. H o w d o e s p H a ff e c t e n z y m e a c t i v i t y ? Enzymes are sensitive to pH changes. Each enzyme has an optimum pH at which its structure and active site are ideal for binding to the substrate. Deviations from this pH can alter t h e e n z y m e ’ s s h a p e a n d r e d u c e i t s e ff e c t i v e n e s s. 3. What is the role of catalase in cells? C a t a l a s e p r o t e c t s c e l l s b y b r e a k i n g d o w n h y d r o g e n p e r ox i d e , a b y - p r o d u c t o f m e t a b o l i c r e a c t i o n s , i n t o w a t e r a n d ox y g e n , p r e v e n t i n g ox i d a t i v e d a m a g e.