CHEM 301 Biochemistry Lab Exercise 1 PDF

CHEM 301: BIOCHEMISTRY | LABORATORY EXERCISE 1: QUALITATIVE TEST FOR PROTEINS PRELIMS S.Y. 2022 – 2023 MARIAN DARA T. TAGOON, MSc OUTLINE all throughout th...

CHEM 301: BIOCHEMISTRY | LABORATORY EXERCISE 1: QUALITATIVE TEST FOR PROTEINS PRELIMS S.Y. 2022 – 2023 MARIAN DARA T. TAGOON, MSc OUTLINE all throughout the I. What Are Proteins? amino acids. The A. Structures of Amino Acids characteristics of the II. How To Test Substances For Presence Of amino acids only vary Proteins? depending on the Side A. Biuret Test Chain (R) group that 1. Biuret Test Results they have. B. Ninhydrin test Amino acid molecules 1. Ninhydrin Test Results undergo condensation C. Hopkins-Cole Test reaction to form a 1. Hopkins-Cole Test Results specific type of linkage D. Sakaguchi Test known as peptide 1. Sakaguchi Test Results linkage. III. Theoretical Data ○ Sources: A. Composition of Different Amino Acids on Plants synthesize proteins from the Different Sample inorganic substances present in B. Expected Results the air and in the soil. IV. Answer to the Post Lab Questions Animals cannot synthesize proteins from such materials. Animals obtain proteins from I. WHAT ARE PROTEINS? plants or from animals which Proteins obtain the same from plants. ○ Large biomolecules that primarily contain C, H, O, and N. A. STRUCTURES OF AMINO ACIDS ○ Some contain nonmetals S, P and I. Nonpolar Amino Acids ○ Others contain metals such as Fe, Cu, ○ The side chains are usually comprise of or Mn. molecules that do not have the ability to ○ Made up of simple molecules - amino form bonds with other hydrogen and acids. oxygen to form water = Insoluble in Amino Acids water. Simplest functional Polar Amino Acids form of protein ○ Have a portion that can bind to either Comes with other hydrogen or oxygen in order to form with benzene rings and the water = Soluble in water. aromatic side chains Acidic Amino Acids (i.e., phenylalanine, ○ Their side chain is (-) negatively tryptophan, and charged. tyrosine). ○ Less soluble in water than basic amino Are molecules acids. containing: Basic Amino Acids ○ Amino (NH2) ○ Their side chain is (+) positively group charged. ○ Carboxylic ○ More soluble in water than acidic (COOH) group amino acids. ○ Side Chain (R) group Amino (NH2) group & Carboxylic (COOH) group are consistent AMANDO & LIM, R. | 2F | 1 of 6  KOH), protein reacts with copper (II) ions to form a violet colored complex called biuret (Rhuman’s purple). (https://www.youtube.com/watch?v=ufec 89A47uM) ○ Copper will actually bind to peptide links resulting in the polypeptides. 1. BIURET TEST RESULTS In the Biuret Test, the only positive test is the color purple. ○ WHY? - The copper sulfate originally got its color blue from copper because copper would exist blue in the natural world. Although there is a color change II. HOW TO TEST SUBSTANCES FOR PRESENCE in the solution (from transparent to blue), OF PROTEINS? the blue coloration does not indicate positive for Biuret test, it only took on the A. BIURET TEST original blue color of copper. Biuret Test Egg Albumin Solution ○ Reagent: Copper Sulfate (CuSO4) ○ Contains a lot of amino acids and is a It would bind to peptides. very big protein = has a positive result in ○ This test is used to detect the presence all tests. Thus, used as a POSITIVE of peptide bonds (DIPEPTIDE BONDS). CONTROL. Dipeptide Bonds ○ Produces a more deep purple color Two peptide bonds compared to milk or glutathione. were formed WHY? - Egg is composed of Usually require three or albumin and albumin is a more amino acids that complex protein. Therefore it group together contains more amino acids and ○ When treated with copper sulphate peptides. solution in presence of alkali (NaOH or AMANDO & LIM, R. | 2F | 2 of 6  ○ NOTE: The color intensity changes Brown color with the complexity of amino acid ○ Positive indicator for arginine that you have in the sample. B. NINHYDRIN TEST Ninhydrin Test ○ Check whether a given analyte contains amines or α-amino acids. For hydroxyproline and proline, a yellow color is obtained. For asparagine, brown color is obtained. No color indicates negative reaction. (https://www.youtube.com/watch?v=Od2 aOyoyM9s) ○ Once you add your Ninhydrin reagent, C. HOPKINS-COLE TEST this would react with the amino acid and This test is used for detecting the presence of it will break the amino acid. tryptophan in protein. In proteins, Positive test HOW? - It will break part of the indicates a purple ring appears between the two carboxyl group and it will lose layers due to the presence of tryptophan. the nitrogen from your amine Negative test shows no appearance of a purple group to form ammonia and ring. carbon dioxide. The carbon (https://www.youtube.com/watch?v=7w3xLNfSb7 dioxide will come from the s) oxygen that was given out by ○ Unlike biuret and ninhydrin tests, the Ninhydrin, making the Hopkins cole test is only specific to Ninhydrin a reduced form detecting tryptophan. Since it detects because it reduced a one tryptophan, that means we can actually negatively charged particle and use this as a confirmatory test for a transferred it to the carbon particular amino acid. dioxide that comes from the ○ Some groups observed that the result of carboxyl group. the Hopkins cole instead of blue It also produces gas. coloration or purple, they observed The reduced Ninhydrin that was yellow coloration. Theoretically, we have created from the initial reaction tryptophan, under the presence of together with the ammonia and sulfuric acid and basically Hopkins-Cole the ninhydrin from the original test is made up of glyoxylic acid.This solution will further react to form glyoxylic acid, reducing the NH2, will the violet blue complex. lead to a purple color product. The reagent itself is no longer working, considering that the reagent has been used by many laboratory classes, and the quality were not checked (pipettes were probably wet when they dipped in the hopkins cole thereby also altering its composition, that's why they did not observed the purple colored product). 1. NINHYDRIN TEST RESULT Purple-colored complex ○ Positive indicator for all alpha amino acids Yellow color ○ Positive indicator for proline, hydroxyproline, and other derivatives AMANDO & LIM, R. | 2F | 3 of 6  1. HOPKINS-COLE TEST RESULT 1. SAKAGUCHI TEST RESULT Purple ring present Red colored complex ○ Hopkin’s Cole POSITIVE test ○ Sakaguchi POSITIVE test ○ Tryptophan is present ○ Presence of arginine or a guanidinium Purple ring absent compound ○ Hopkin’s Cole NEGATIVE test Red colored complex absent ○ Tryptophan is absent ○ Sakaguchi NEGATIVE test ○ Absence of arginine or a guanidinium compound D. SAKAGUCHI TEST Sakaguchi Test E. XANTHOPROTEIC TEST ○ The test is a specific test for arginine Xanthoproteic Test where the guanidinium group of arginine ○ Used for detection of amino acids reacts with 1-naphthol or 𝝰-naphthol to containing phenolic or indolic groups like produce a colored product. Reaction phenylalanine, tyrosine, and results in the formation of a red-colored tryptophan(aromatic amino acids). The complex due to the formation of an test is named Xanthoproteic test due to indole-like structure. the formation of a yellow precipitate of (https://www.youtube.com/watch?v=Hjlo xanthoproteic acid. ozBunXE) (https://www.youtube.com/watch?v=eWF ○ Many laboratory experiments, the alpha D-B7fwZY) naphthol is originally transparent or ○ Phenolic or the benzene rings which is cloudy white color. What we used in the basically made up of 3 types of amino laboratory was molisch reagent, and this acid (phenylalanine, tyrosine and molisch reagent already contains a dye. tryptophan). What happens is that they Technically it is challenging when we utilize these rings from the tyrosine as a have observed the coloration of the site of attachment for the O2N complex. reddish. The thing that we did was to The O2N complex is basically test or look at the test tube against the [bis(4-methoxyphenyl)methyl]carbamoyl} light, and then we tried to inspect what benzyl). But if there’s no ring, particularly color it had. it would attach to the fifth ring of the ○ Alpha naphthol will react to the aromatic structure, and there will be no guanidium group such that naphthol will site of attachment for the O2N complex. attach to the one of the parts of So it will only do that at high guanidium part,it would lose its concentrated nitric acid, when we hydrogen and it will replace of a subjected it further to basic pH, from bromine. acid we also added NaOH in order to observe the precipitation, there was precipitation formed but very faint. That can also be attributed to basically the amount of the amino acid present in the solution. AMANDO & LIM, R. | 2F | 4 of 6  B. EXPECTED RESULTS Biuret Test ○ Since there are 2 amino acids that compose the aspartame, therefore, we do not see any coloration because the phenylalanine and aspartic acid only produced one peptide bond ○ It is an indication of peptide as long as it detects the dipeptide bonds 1. SAKAGUCHI TEST RESULT ○ Aspartame can form 1 bond therefore, it Presence of the Dark yellow or orange color is negative ○ Xanthoproteic test POSITIVE ○ Glutathione, Egg white and milk should ○ Presence of aromatic amino acids have a positive reaction to it because (tyrosine and tryptophan) they already formed the minimum Absence of the dark yellow or orange color requirement of 2 peptide bonds. ○ Xanthoproteic test NEGATIVE Ninhydrin Test ○ Absence of aromatic amino acids ○ It detects all types of alpha amino acids, (tyrosine and tryptophan) and since all of these sample contain amino acids, therefore it should be positive for the ninhydrin test Hopkins Cole Test ○ We should not observe any changes on the aspartame and glutathione because it has no tryptophan. On the other side, we should be able to see any positive reaction when it comes to egg white and the milk. Sakaguchi Test ○ No changes in aspartame and glutathione because they dont have III. THEORETICAL DATA arginine but in egg white and milk we should be able to see the light red color. Xanthoproteic Test ○ There should be a yellowing of aspartame, egg white and milk considering that they do contain the phenylalanine, tryptophan or tyrosine. IV. ANSWER TO THE POST LAB QUESTIONS 1. What is the principle behind each test? Give general chemical equation? 2. What group of amino acids is identified in each test? 1. NINHYDRIN TEST RESULT1. NINHYDRIN TEST R 3. Account for the difference in color intensity A. COMPOSITION OF DIFFERENT AMINO ACIDS between each sample (if there are any). ON THE DIFFERENT SAMPLE a. Intensity of the reagent, Amount of Aspartame is made up of phenylalanine and protein/amino acid/peptide aspartic acid i. That means the color intensity Glutathione is made of cysteine, glutamic acid, does change and it is brought and glycine about by the amount of protein Egg white solution contains almost all amino amino acid or peptide that is acids present in the sample. Milk which contains lysine, histidine, leucine, ii. Take Note: valine, tryptophan, arginine 1. The intensity of the color change will not give you a quantitative picture on how much AMANDO & LIM, R. | 2F | 5 of 6  amino acid protein or peptide you have. It will only tell the particular solution has more amino acid, peptide or protein but it will not tell you how much, or the concentration of the amino acid because that is the limitation of the qualitative test. 4. Are all the proteins positive for all tests? Why or why not? b. It will not because there were no universal tests in order to indicate the presence of all proteins. Another reason is that the different test that we performed might have some problems with the reagent therefore, we didn’t get the expected results. AMANDO & LIM, R. | 2F | 6 of 6  CHEM 301: BIOCHEMISTRY | LABORATORY EXERCISE 2: PROTEIN DENATURATION PRELIMS S.Y. 2022 – 2023 MARIAN DARA T. TAGOON, MSc OUTLINE B. THE FORCES THAT HOLDS PROTEINS TOGETHER I. Protein Denaturation Ionic bond A. How Proteins are Formed ○ opposite charges in the hydrophobic B. The forces that holds protein together core of proteins. C. Expected Observations of the The bonds that result from Experiment electrostatics attraction between D. Post lab Questions the 2 different charges of proteins or side chain of amino I. PROTEIN DENATURATION To observe the effects of several denaturing acid. Basically, it forms a reagents on a protein sample stronger connection because it To differentiate the effect of these denaturing is electrostatic attraction, you reagents to the protein can see on the secondary sample structure. To cite practical applications of these denaturing Peptide bond agents. ○ covalent bond to create chains of peptides. A. HOW PROTEINS ARE FORMED First things first covalent bonds Proteins are formed by basically, there are 4 which is theThis would be the structures of your proteins. bonds that would hold the You have the most important structure, you amino acids together. have the primary structure, the chains of your Hydrophobic bond amino acids which are also called polypeptides. ○ reaction between nonpolar molecules. Primary structure is held together with ionic Which is the reaction between bonds and covalent bonds between your nonpolar molecules, so you peptides. have nonpolar molecules we Secondary Structure is the folding of your called them; they are molecules protein in a 2D manner,which is alpha-helix or that do not have the ability for O beta-pleated sheet. This is held together by or H to attach to form water. different types of bonds: ionic bonds, disulfide In most cases, hydrophobic bonds which would be your covalent bond, interactions form the hydrophobic bonds,and etc. alpha-helix, the secondary Tertiary structure the further folding of structure and some parts of secondary structure. If the secondary has only tertiary structure. one folding pattern, in the tertiary it is the 3D Their formation, since they are folding pattern of your protein. hydrophobic they tend to form Furthermore, Quaternary structure is the inside the proteins or they tend assembled units of different types of tertiary to fold towards the inside of the structure, creating a more complex protein. protein, so that they will not be exposed to water or aqueous environment. Hydrogen bond ○ Hydrogen bonding affects the tertiary structure of a protein. Very weak bonding. Basically, what makes up your tertiary structure of the proteins. This is a very weak bond. PAULINIO & TABINAS | 2F | 1 of 4  Tertiary and quaternary This makes heat a poor structure largely depend on the denaturing agent because the hydrogen bond and they are the protein still has the ability to easiest to break apart. renature itself. When we say Disulfide linkage renature, it will re-align to its ○ Formed between cysteine residues in original structure. peptides and proteins. If the temperature goes beyond Cysteine only has a sulfhydryl 70 degrees Celsius and more, group. the denaturation is irreversible. Dispersion forces ○ Alcohol Nonpolar atoms become polar due to Alcohol also disrupts the uneven distribution of electrons. hydrogen bonding with the Tend to become polar due to protein uneven distribution of electrons, Unlike with heat that increases so that can also happen within its kinetic energy to lose the the protein structure. hydrogen bond. Alcohol is R-OH. The OH group will bind C. EXPECTED OBSERVATION OF THE EXPERIMENT with the amino acid residues. During the experiment, we tried 2 Denaturing The amino acid residues agents combine with each other, ○ Physical Denaturing agent represented instead na sila ang mag by heat combine, the OH of alcohol ang Used heat papasok. So therefore, if si OH Breaks the bond of hydrogen ang papasok, walang mag bond and the dispersion forces foform. So may isang acid ○ Chemical Denaturing agent represented residue or amino acid residue by organic chemicals na blangko. So this breaks the e.g. alcohol, acids & bases, structure of your protein. heavy metals, alkaloidal In the observation, you solutions observed that 70% of alcohol Expected Observation of the Experiment actually produces more ○ Heat denaturation than your 95%. If you apply heat, it actually Although, it’s a bit contradictory increases the kinetic energy of because many of the references the protein molecules. So when would indicate that the extent of we say kinetic energy, The denaturation also increases as molecules will have the ability to alcohol concentration increases. move or vibrate caused by the However, since your 95% heat or UV radiation. Once it alcohol evaporates faster, it vibrates, it weakens the leaves a little time for the cell to hydrogen bonding. be exposed to the alcohol. So Now if we have to recall, even if this is the reason why you are the hydrogen bond breaks able to observe more because of the radiation/ heat denaturation on your protein by kay naga move parin sya. So it the 70% alcohol. basically breaks the bond. So ○ Strong Mineral Acids therefore kung ma break nya Change in pH of the ang bond, oxygen will still have environment where your protein a negative charge and the is suspended can disrupt the hydrogen will still have a south bridges such as your positive charge. covalent bonds and hydrogen Since they have opposite bonding that is formed between charges, they will be attracted the amino acids side chains. to each other. Since na break The precipitation of the product ang structure, the bond can still would actually vary. reconnect with one another Example: If you use acid to after heating. denature protein, your scid will PAULINIO & TABINAS | 2F | 2 of 4  lead to the precipitation of your the secondary and some parts protein, but this precipitation of the primary structure. becomes less soluble. This is the reason why there is more DENATURING AGENTS OBSERVATIONS precipitation sa acid compared when using sabon or any OH. Heat and radiation supplies Sa sabon, you observed that it kinetic energy to protein was very soluble, it is because HEAT molecules, causing their of your NaOH or if you have a atoms to vibrate more rapidly high pH, your protein becomes and disrupting relatively weak hydrogen bonding and soluble. This is the reason why dispersion forces. Irreversible you did not observe a lot of denaturation at higher precipitation on your protein temperature. when you NaOH, compared to when you use nitric acid. Disrupts intramolecular ○ Heavy Metals hydrogen bonding within the Whether you are using silver protein by competing with the amino acid residues from nitrate or copper sulfate, the forming new hydrogen bond. heavy metal would lead to the Breaks the tertiary structure disruption of the ionic bonds of the protein. and also your disulfide linkages, ALCOHOL particularly for the cystine Extent of denaturation group. increases as alcohol In this case, the silver nitrate concentration increases (Nikolaidis et al., 2018). would form a precipitation Evident via precipitation. because the albumin and the However, since 95% alcohol silver react to form a silver evaporates faster, it leaves albuminate, which is an little time for the cell to be insoluble product. exposed to the alcohol However, the copper sulfate still has the same reaction that change the environmental pH of proteins, which disrupts the disrupts the disulfide cysteine salt bridges and hydrogen bonds. Since copper sulfate has bonding formed between the an alkaline pH. side chains Alkaline pH is usually STRONG MINERALS maging soluble protein NaOH – Protein is soluble under alkaline with strong alkali HNO3 - white precipitate ring conditions. is obtained. Protein becomes So this is the reason why, less soluble. between silver nitrate and copper sulfate. We did not These ions form strong bonds observe any precipitate with the with the carboxylate anions of silver nitrate compared with the the acidic amino acids or SH copper sulfate. Since copper groups of cysteine, disrupting ionic bonds and disulfide sulfate is alkaline. linkages. ○ Alkaloidal Reagents HEAVY METALS We used tannins or tannic AgNO3 – white precipitate acids and picric acid obtained from the formation of The tannic and picric acids silver albuminate therefore disrupt the ionic bonds. albumin is precipitated by Between the heavy metals and heavy metal ions. all the chemical denaturing CuSO4 - disrupt disulfide agents that we use, heavy Cystine bonds, binding to metals and the alkaloidal individual sulfhydryl groups. reagents gives more damage Alkaline thus protein is slightly because it breaks the ionic soluble bonds which technically holds PAULINIO & TABINAS | 2F | 3 of 4  The egg is called a These reagents combine with positively charged amino “chelating agent” groups in proteins to disrupt because it collects all ALKALOIDAL ionic bonds. the compounds REAGENTS together so that it can Tannins - are water-soluble immobilize the phenolic compounds with the compound by reducing ability to precipitate proteins the toxicity. from aqueous solution. Picric acid - Yellow precipitate is obtained D. POST LAB QUESTIONS Give ONE practical application of each agent presented. ○ Heat food/cooking ○ Acid cooking (e.g. kinilaw, atsara, food preservation Poisoning Home remedy of suspected poisoning The home remedy is ipakain sa tao na is suspected to be poisoned ng egg white The egg albumin contains - ion, and the - ion would react with the + ion particularly if it is heavy metal poisoning. Example is silver nitrate, it would react to albumin, resulting in silver albuminate. Instead na ang atake sa heavy metal is sa body natin, it reduces toxicity because the negative ions of the egg albumin would react to the positive charge of the heavy metal. Moreover, metals will undergo a redox reaction with the egg albumin to form a complex. Once it forms a complex, basically the toxicity of the the poison will reduce because instead na sa cells ng ating stomach mag atake, it will attach on the egg PAULINIO & TABINAS | 2F | 4 of 4  CHEM 301: BIOCHEMISTRY | LABORATORY EXPERIMENT 3: ENZYMES PRELIMS | S.Y. 2022 – 2023 MARIAN DARA TAGOON OUTLINE I. Enzymes A. Catabolism vs Anabolism B. The Role of Enzymes C. Mechanism of Enzymatic Action D. Enzyme Components E. Factors Affecting Enzymatic Activity F. Theoretical Data G. Post Lab Questions H. Enzymes Structures 1. Amylase 2. Pepsin 3. Catalase I. ENZYMES We used 3 types of enzymes: Pepsin, Amylase, and catalase All of these enzymes are found in our body. ○ So for the amylase is found in our saliva for the initial chemical breakdown of your carbohydrates ○ We also have pepsin found in our stomach that breaks down your protein ○ Lastly, the catalase is found all over our body to neutralize your hydrogen peroxide that is basically harmful well, it is a by-product of respiration but nevertheless it’s a harmful by-product and para ma-neutralize siya in order for B. THE ROLE OF ENZYMES our survival the catalase is there to work Catalysts - substances that speed up chemical against it. reaction without being used up in the reaction by lowering activation energy A. CATABOLISM VS ANABOLISM Enzymes are biological catalysts (e.g. sucrase - Catabolic reactions provide building blocks for enzyme that breaks down sucrose to fructose anabolic reactions and provide energy to drive and glucose) anabolic reactions Enzymes are catalysts. Catalysts can be The buildup of molecules like anabolism anything, it can be organic or inorganic in nature The breakdown of molecules like catabolism but again your enzymes is a te of organic or All metabolic functions within the organism, the biological catalyst. human body, or even the bacteria would always Your reactions can run without the presence of use enzyme or would rely on enzyme for the enzyme but it usually takes a longer time and the transformation of molecules into different activation energy is also very much high, its a products. very expensive process without the enzyme, thereby it also produces heat which can also lead to the denaturation of the protein kase this activation energy also releases a lot of heat. DOTIMAS & BAQUIAL | 2F | 1 of 4  E. FACTORS AFFECTING ENZYMATIC ACTIVITY Temperature- the higher the temperature, the faster the enzymatic activity but only up to optimal temperature (35 to 40 degrees celsius). Always remember the higher the temperature, the faster the enzymatic activity but only up to certain point. Kasi ang enzymatic activity based on temperature has an optimal range which is 35 to 40 degrees celsius pH- extreme pH can cause enzymes to denature. Although in some cases yung enzymes require u slow pH such as the case of your pepsin kailangan niya talaga ng atleast two na pH level in order to work properly. Substance concentration- higher substrate, C. MECHANISM OF ENZYMATIC ACTION higher chemical reaction (but should have Lock and Key Mechanism enough active sites) ○ Substrate binds to the active site Inhibitors- can fill active site of the enzyme (substrate-specific) (competitive inhibitor) or they interact with ○ Formation of enzyme-substrate complex another part of the enzyme (allosteri inhibition) ○ Substrate is transformed into product changin the shape of the enzyme making it ○ Product is released and enzyme is non-functional. unchanged. F. THEORETICAL DATA D. ENZYME COMPONENTS Apoenzyme - protein portion of the enzyme Cofactor - non-protein component (e.g. Calcium, Zinc, Magnesium) ○ Coenzyme - cofactor made from organic molecule (e.g. Vitamin B), Riboflavin, Niacin, Folic Acid, Vitamin E, Vitamin K) Pepsin Apozyme or apoenzyme which is the inactive ○ The pepsin + HCl could have inhibitory form it becomes active when you have a cofactor effects because more denaturation was or coenzyme attach to it, to lead or that would observed on the pepsin only tube than result to the holoenzyme and once meron ng the pepsin + HCl tube. holoenzyme this would lead to the activation of Amylase the active site where substrate can attach itself. ○ The addition of iodine detects the presence of starch. Iodine A detector of starch. ○ Once iodine detects starch, it would turn blue or purple, black or green, something that is along dark colors. ○ In the starch + saliva, we are assuming that after 15 minutes immersed in a water bath, the starch should’ve been broken down into simpler glucose units by the saliva. So, once you add iodine, DOTIMAS & BAQUIAL | 2F | 2 of 4  the color of the solution must present the ○ Pepsin - endopeptidase that breaks color of iodine. However, some cases down protein with water (hydrolase) tested positive in the iodine test for the ○ Amylase - hydrolase (act on the α (1→4) starch + saliva tube. This may mean glycosidic linkages in glycogen, starch, that: and related α-glucans) that needs alkali The enzymatic activity is slower, environment so we could have increased the ○ Catalase - Oxidoreductase (it has the incubation period. Instead of 15 ability to donate or receive electron for minutes, let’s try to see if we the byproduct) see changes after 25-30 Explain the mechanism of enzyme action on its minutes. substrate presented in the experiment. Identify The saliva donor ate before the substrate and the products of each digestion he/she donated the saliva. So, performed. that means all of the amylase or Explain the specificity of enzymes on its all of the enzymes are still substrate. occupied in their active site or the starch has occupied the H. ENZYME STRUCTURES active site of the amylase. Catalase 1. AMYLASE ○ It neutralizes effects of H2O2 - a Amylase reactive oxygen species (ROS) which is ○ Starch is made up of chains of glucose one of the causes of oxidative stress in Glucose our cells. A simple sugar ○ Sodium Bicarbonate Becomes an insoluble Presence of bubbles in the fiber when arranged in sodium carbonate solution chains doesn’t mean that the sodium ○ Starch is being cut by the amylase in the bicarbonate is being glycosidic linkages, resulting in metabolized by your catalase. individual units of glucose. This is what WHY?: Catalase is very specific happens after you heat up the amylase to the substrate, H2O2. The + starch. It releases starch as a simple formation of bubbles is due to glucose, which is a simple sugar. the nature of the sodium bicarbonate of being a bicarbonate (a carbonic acid). It will react to the potato which has another pH level. Therefore, it will produce bubbles. It reacted to the pH level of the potato that’s why it created bubbles. It is still a negative reaction because the catalase is not the one that produces bubbles, rather it is the sodium bicarbonate reacting to the pH of the potato. ○ Acetic Acid 2. PEPSIN Slight bubbling is observed due Pepsin to live potato cells at the bottom ○ Swirly structure indicates plenty of of the test tube. attached linkages or amino acids to form the protein. G. POST LAB QUESTIONS ○ With the presence of pepsin at a very What group of enzymes does each enzyme in low pH, it will cut off the amino and the experiment belong? What is its specific carboxyl group. function? DOTIMAS & BAQUIAL | 2F | 3 of 4  3. CATALASE Catalase ○ Will metabolize H2O2 into water and oxygen. DOTIMAS & BAQUIAL | 2F | 4 of 4