Hydrolysis of Intact Protein
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c Christine Umali, Jon Lester Uy, , Jose Maria Veloso, Norjem Villanueva and Jaquelyn Wodi Group 9 2A Medical Technology Biochemistry Laboratory
Proteins are polymers of amino acids and it is the most abundant organic molecules found in living cells. Myoglobin is an iron- and oxygen-binding protein found in the muscle tissue of vertebrates in general and in almost all mammals. This portion of the experiment refers to the denaturation of intact myoglobin through hydrolysis. The pre-isolated myoglobin from meat sample was subjected to acid, alkaline and enzymatic hydrolysis to disrupt its native conformation and thus making a suitable hydrolysate for qualitative and quantitative characterization. Both acid and alkaline hydrolysis involved autoclaving the mixture at a specific pressure and neutralizing by their counterparts. In acid hydrolysis, HCl which served as an acid medium was used to denature the protein and NaOH was used to neutralize it. Consequently, NaOH was used as a basic medium in alkaline hydrolysis and HCl was used to neutralize the mixture. In enzymatic hydrolysis, however, a protease solution was used as a medium and the mixture was incubated in a water bath for a specific temperature. By the end of this portion of the experiment, the appearance of each of the hydrolysates produced by the three hydrolysis procedure was noted. The types of hydrolysis were also compared according to their efficiency in hydrolyzing proteins.
Proteins are a class of organic compounds which are present in and vital to every living cell. Proteins hold together, protect, and provide structure to the body of a multi-celled organism; they catalyze, regulate, and protect the body chemistry and in the form of hemoglobin, myoglobin and various lipoproteins, they affect the transport of oxygen and other substances within an organism. Myoglobin is a singlechain globular protein of 153 or 154 amino acids, containing a heme which is an ironcontaining porphyrin prosthetic group in the center around which the remaining apoprotein folds. Biologically, active proteins, like myoglobin, are made up of polymers consisting of amino acids linked by covalent peptide bonds. These bonds are broken when the protein undergoes hydrolysis. In hydrolysis, the protein is subjected to extreme conditions usually at high temperatures by prolonged boiling in a strong acid or strong base or using an enzyme such as the pancreatic protease enzyme to stimulate the naturally occurring hydrolytic process. This will cause denaturation of the protein meaning that the protein¶s conformation is altered by the breaking of peptide bonds. This results to a solution containing amino acid fragments which is then called the hydrolysate. Denaturation alters protein function, demonstrating a relationship between structure and function. Hydrolysis of protein and analysis of products are done to obtain information about their compositions. The three most common types of hydrolysis are aid, basic and enzymatic hydrolysis. Acid hydrolysis implies a chemical mechanism of hydrolysis catalyzed by a Brønsted or Arrhenius acid. By contrast, it does
not usually imply hydrolysis by direct electrophilic attack²as may originate from a Lewis acid. Nevertheless, the type of hydrolysis carried out with a basic medium is termed basic hydrolysis. Lastly, an enzymatic hydrolysis is by the addition of specific enzymes called proteolytic enzymes or simply proteases which refer to a group of enzymes each to hydrolyse specific peptide bonds of proteins. This portion of the experiment aims to perform acid, alkaline, and enzymatic hydrolysis on the isolated proteins and enumerate the advantage and disadvantages of each type of hydrolysis.
? ! Isolated gluten, casein, albumin and myoglobin, 6M HCL, 4M NaOH, 1M HCl, 1M NaOH, saturated protease solution and red and blue litmus paper ? "#! " $? #%!c&!"&%'()#) ")%( For this type of hydrolysis, 5 ml of the 6M HCl was added to 0.5 g of the isolated myoglobin in a hard glass test tube. The test tube was properly labeled according to the format given. Then cotton was placed to stopper the tube before it was subjected to autoclaving at 15 psi for 5 hours. Alternatively, the protein sample could be placed in a sealed container containing 6M HCl. The whole container is then placed in a microwave oven for about 5-30 minutes with temperatures up to 200oC. This will vaporize the 6M HCl which will come in contact with myoglobin and hydrolyse it. Then, the appearance of the mixture was noted after autoclaving. Then, distilled water with a volume of 10 mL was added and the mixture was transferred into a 250-mL
beaker. The mixture was neutralized with 1M NaOH and that neutralized mixture was used for characterization tests and chromatography. *? +%(c&!"&%'()#) ")%( In alkaline hydrolysis, 10 mL of 4M NaOH was added to 0.5 g isolated myoglobin in a hard glass test tube. The test tube was properly labeled according to the format given. Like in acid hydrolysis, cotton was placed to stopper the tube before it was subjected to autoclaving at 15 psi for 5 hours. Also, the appearance of the mixture was considered and noted. Then again, distilled water with a volume of 10 mL was added in the mixture then was transferred into a 250-mL beaker. Nevertheless, the mixture was neutralized with 1M HCl and that neutralized mixture was used for characterization tests and chromatography. s? (,&)%# c&!"&% ' ()#)
")%( To perform the enzymatic hydrolysis, a 1g/100 mL distilled water protein mixture was prepared. A volume of 10 mL of the protein mixture and 10 mL of the saturated protease was mixed. Alternatively, a 0.050 g of protease may be added directly to 50 mL protein mixture. Then, a 10mL 0.1 M phosphate buffer with a pH of 7.5 was added. The tube was incubated in a water bath maintained at 35-40 oC for about an hour. Again, alternatively, digestion may be carried out overnight at room temperature. Then, the mixture was allowed to cool and was then used for qualitative procedures for color reactions and chromatography.
Before performing hydrolysis,
the protein myoglobin was first isolated from a meat sample. After the isolation process, the intact protein appeared to have a pale red turbid solution. This intact myoglobin was subjected to hydrolysis basically to break down the protein into smaller amino acid fragments which will make the qualitative and quantitative determination of its components easier. Table 1 shows the appearance of each of the hydrolysates collected from each of the three types of hydrolysis.
- $ %) ' ) "(# ' ) c&!"&) !' c&!"&% Acidic Basic Enzymatic
#"%)%(')c&!"&) Dark brown solution Clear solution Dark brick red solution with no turbidity
In acidic hydrolysis the isolated myoglobin the 6M HCl served as the medium for hydrolysis. In other words, it is the HCl that serves as the catalyst for the denaturation of myoglobin. The mixture was then subjected to autoclaving or it could either been put into a microwave at high temperatures. The autoclave machine provides an environment with a pressure of 15 psi (pound per square inch) enough to cause denaturation of the protein if done in a sufficient amount of time. Alternatively, if the isolated myoglobin was put in a microwave containing 6M of HCl and with a temperature of 200oC, it would supply the mixture a temperature which is suitable for denaturation as well. The acid catalyst which is the HCl will vaporize and will eventually react with the protein, thus hastening the hydrolysis. From a pale red turbid solution, the hydrosylate appeared to be a dark brown solution after autoclaving as shown in Table 1. Then, the mixture was neutralized by 1M NaOH to make an appropriate solution for the quantitative and qualitative reactions. This neutralization will cause the denaturation of myoglobin because the acid-base interaction will form a salt bridge that disrupts the protein¶s structure. On the other hand, the basic hydrolysis¶ procedure is almost the same as the acidic hydrolysis. And so, like in acidic hydrolysis, the isolated myoglobin was subjected to an environment with the same pressure of 15 psi sufficient to hydrolyse the protein. The only difference is that it uses a basic catalyst in the presence of 4M NaOH and unlike in acidic hydrolysis, the basic hydrolysate was neutralized with 1M HCl which is acidic. This makes sense since neutralization is the reaction between an acid and a base that forms salt. In protein denaturation, the salt formed in the neutralization will alter the conformation of the protein by further straitening the protein chain. Based on Table 1 from a pale red turbid solution, the protein mixture turned into a clear solution, which thus indicated an alteration in its conformation as well. This means that the myoglobin can be undergo denaturation with a basic catalyst as well.
In enzymatic hydrolysis, a type of special enzyme was used to hasten the denaturation process of myoglobin under high temperatures. The principle behind the type of enzymatic hydrolysis done is the same as the mechanism that takes place in the human body. Enzymes are the body¶s natural catalysts. The catalytic action of enzymes allows the hydrolysis of proteins, fats, oils, and carbohydrates. As an example, one may consider proteases which are enzymes that aid digestion by causing hydrolysis of peptide bonds in proteins. They catalyse the hydrolysis of interior peptide bonds in peptide chains, as opposed to exopeptidases which is another class of enzymes that catalyse the hydrolysis of terminal peptide bonds, liberating one free amino acid at a time. In the experiment, the isolated myoglobin was put in an environment which is almost the same as the environment inside the human body; with a pH of 7.5, a temperature of 35-40 oC and with an enzyme catalyst. And so, as expected, the protein was denatured as proved by the change in appearance of the sample from a pale red turbid solution to a dark brick red solution without turbidity as shown in Table 1. The hydrolysate does not need to be neutralized since it is neither basic nor acidic, thus, is not susceptible to other reactions after it cools.
and tertiary structure are disrupted. Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions. This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted. Salt bridges result from the neutralization of an acid and amine on side chains. The denaturation reaction on the salt bridge by the addition of an acid results in a further straightening effect on the protein chain. Since denaturation affects the protein¶s structure, it could also affect its function. Denatured myoglobin will not be able to carry oxygen since its structure which is a vital part of this function is disrupted. Among all the three types of hydrolysis, the acid and alkaline hydrolysis are better than enzymatic. This is because proteases, which are commonly used in enzymatic hydrolysis, do not catalyze the hydrolysis of all kinds of proteins. Their action is stereo-selective: Only proteins with a certain tertiary structure will be targeted. As some kind of orienting force is needed to place the amide group in the proper position for catalysis. The necessary contacts between an enzyme and its substrates (proteins) are created because the enzyme folds in such a way as to form a crevice into which the substrate fits; the crevice also contains the catalytic groups. Therefore, proteins that do not fit into the crevice will not undergo hydrolysis. This specificity preserves the integrity of other proteins such as hormones, and therefore the biological system continues to function normally.
Campbell, M. K., (2012./2009) u . China: Brooks/ Cole, Cengage Learning Crisostomo, C.C., Daya, M. L., et al (2010).
u . Quezon City: C & E Publishing http://goldbook.iupac.org/H02902.html %. "$)" #) "'&.-%( Over all, it could be deduced that proteins can undergo denaturation in high levels of pressure temperature. And because hydrolysis breaks peptide bonds in protein, it could cause a drastic change in the protein¶s conformation and structure which eventually leads to denaturation. Figure 1 shows the original structure of myoglobin. Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides)
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