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Isolation and Characterization of Proteins (Acid Hydrolysis of Myoglobin) 2GPh Group 7 Lourice Sales*, Hannah Seron, Alianna Serrano,

Karissa Sison, Marjorie So, Teng Tsai

ABSTRACT Myoglobin is a protein in the heart and skeletal muscles that has oxygen attached to it. It is extracted and hydrolyzed by acid and subjected to different qualitative color tests. In this experiment Myoglobin has been extracted from raw beef and as it was subjected to various qualitative color tests positive results were seen in Fohl s and Sakaguchi tests. This means that proteins and amino acids are present in the solution extracted from the beef.

Introduction Proteins are probably the most important class of biochemical molecules, although lipids and carbohydrates are also essential for life. They are the basis for the major structural components of animal and human tissue. Proteins are natural polymer molecules consisting of amino acid units. The number of amino acids in proteins may range from two to several thousands. Myoglobin is a kind of protein found in the muscle tissue of vertebrates. Myoglobin and hemoglobin are hemeproteins whose physiological importance is principally related to their ability to bind molecular oxygen. Myoglobin is a monomeric heme protein found mainly in muscle tissue where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation oxymyoglobin releases its bound oxygen, which is then used for metabolic purposes. The hydrolysis of amides under acidic condition is basically the same as protein hydrolysis, because peptide bond link is the same as an amide link. If you scale this up to a polypeptide chain, each of the

peptide links will be broken in exactly the same way. That means it will end up in a mixture of the amino acids that constitute the protein. Protein hydrolysates can be tested qualitatively by different reagents and as well as paper chromatography. Partition paper chromatography is widely employed for the determination and separation of amino acids. The solvent migrates along a strip of paper and carries amino acid dissolved in it. Experimental Acid Hydrolysis of Intact Protein Myoglobin extracted from ground beef was hydrolyzed by acid after it was tested with different qualitative color tests. The hydrolysis p r o c e s s undergone by adding 5 mL o f 8 S ulfuric acid to 0.5 grams of intact protein and then autoclaved for 5 hours (15 psi), T10 mL of d i s t i l l e d water was added to the autoclaved sample and then transferred to a 250 m L beaker. After the sample was transferred, 5 mL o f 8 N b a r i u m h yd r o x i d e w a s ad d e d a n d t h e n mixed. The sample was neutralized by adding8N barium hydroxide or 8N sulfuric acid. It was t h e n t e s t e d wi t h

red and blue litmus p ap er check if it was already neutral. sample was filtered and the filtrate collected for characterization test paper chromatography. Qualitative Color Reactions

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The myoglobi n h yd r o l y s a t e s w e r e t e s t e d wi t h diff er ent char act er iz ati on re age nts n amely: B i u r e t , N i n h y d r i n , X a n t h r o p r o t e i c , M i l l o n , Hopkins -Cole, Sakaguchi, Fohl, and Diazo tests. Eight test tubes were prepared for each of thetest reaction. Each test tube consisted of 1 mL of distilled water added to 0.5 mL of hydrolyzed samples. In Biuret test 10 drops of the Biuret reagent was added to one test tube, shaken and the observed for color changes. For Ninhydrin t est, 6- 10 d ro p s of 0.1% N i n h y d r i n s o l u t i o n was added to the dilute sample and heated in a w a t e r b at h . T h e a p p e a r a n c e o f a b l u e v i o l e t coloration was taken note of. In Xanthroproteic test, concentrated nitric acid and concentrated sodium hyd roxid e 10 d rop s each were add ed s l o w l y a n d t h e n mixed. Color changes after each addition were observed. Five drops of Millon s reagent was added to the diluted s amp l e w i t h t he color taken note of. For Hop kin s -C ole t est, 20 d rop s of H opkins -Cole reag ent was s l o w l y ad d e d t o t h e s a m p l e a n d m i x e d w e l l . S u l f u ri c acid a b o u t 2 0 d r o p s w a s slowly added along side the inclined test tube. The color of t he interf ace was t ak e n n ot e of . After the addition of 10 drops of Sakaguchi reagent and mixed,it was let stand for

3 mi n u t e s a n d 3 d r o p s o f 2 % h yp o b ro m i t e w a s a d d e d . A r e d color ind icated t he p res en ce of arginine in the protein. The addition of 20 dropsof 6M sodium hydroxide and a few crystals of l e a d a c e t a t e t o t h e d i l u t e d s a m p l e s p r o vi d e d d a r k brown sediment during b o i l i n g o f t h e s a m p l e p ro v i d e d the p ositiv e result in Fohl s reaction. Lastly, a preparation of 3-5 drops of 1 % s u l f o s a l i c y l i c acid with 3 drops of 5% sodium nitrite was added to 5 drops of the diluted sample and 5 drops of 10% sodium carbonat e solution. A red c o l o r a t i o n w a s observed. Results and Discussion Qualitative Color Test for Hydrolyzed Myoglobin

*Results may have been incorrect because the protein was contaminated Only the Fohl s and Sakaguchi test gave a positive result for the color characterization of the hydrolyzed myoglobin which indicates the presence of arginine, cysteine, and methionine. On the other hand compared with the results of the characterization test of the intact myoglobin, only the Ninhydrin test showed a negative result indicating the absence of an alpha amino acid in the intact protein.

Discussion Xanthroproteic reaction is specific for cyclic a m i n o a c i d s s u c h as phen ylal anin e, t yrosi n e, tryptophan and histidine. Aromatic amino acids r e a c t w i t h n i t r i c acid, yielding a yellow nitro-compound, which chan ges color to orange i n alkaline m e d i u m o wi n g t o t h e f o r m a t i o n o f salt positive for the basic hydrolysis. Si n c e h i s t i d i n e a n d t r yp t o p h a n are aromatic amino acids, Xanthroproteic reaction yielded a positive result for the intact protein but not for the hydrolysate caused probably by the errors or loss of protein during hydrolyzation process. Millon s reagent i s u s e d f o r t h e determination of tyrosine content in proteins. It i s comp osed of s alts of mercur y d i s s o l v e d i n nitric acid. Tyrosine (both free and constitutive of proteins) reacts with reagent and produces a purple-red salt of mercury. Diazo reaction is t y p i c a l f o r p r o t e i n s containing cyclic amino acids such as tyrosine and hi stidin e. The amin o acid s p r o d u c e a r ed c o l o r e d d i a z o d y e i n r e a c t i o n wi t h t h e d i a z o reag ent and the intensity of the color depends on the amount of cyclic amino acid present. I n the intact myoglobin, the diazo r e a c t i o n provided a positive result while negative for the hydrolyzed myoglobin caused probable by the same errors and reas on of the m i l l o n s a n d xanthropeoteic reaction. Sakaguchi reaction is typical for arginine only. Arginine reacts with n a p h t o l a n d produces a red

colored derivative. The presence o f ar ginin e i s p ositiv e f or b ot h the intact and hyd rolyzed m y o g l o b i n a n d s u p p o rt e d b y t h e p r e s e n c e o f a r g i n i n e i n t h e p a p e r chromatography for the hydrolyzed protein. Fohl s reaction is used for determination of S-containing amino acids. Heating of myoglobin sol ution in an alkaline medium leads to the formation of sodium sulfide if the protein contained sulfur amino acids such as cystein e, cystine, or methionine. In further reaction of sodium sulfide with lead acetate a dark brown colored precipitate is formed. F o h l s test is both positive for i n t a c t a n d hydrolyzed myoglobin indicating the presence of c y s t e i n e i n t h e p r o t ei n a l t h o u g h t h e r e s u l t o f paper chromatography yielded a negative result for the hydrolyzed myoglobin. References 1. Crisostomo, A., et al Laboratory Manual in General Biochemistry Quezon City: C & E Publishing, Inc. 2. Acid Hydrolysis. Retrieved January 15, 2011, from http://medicaldictionary.thefreedictionary. com/Acid+Hydrolysis 3. Amino Acids. Retrieved January 15, 2011, from http://en.wikipedia.org/wik i/Amino_acid

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