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BIOCHEM 1 L – Experiment # 05 – 2A BC – Group 2 ISOLATION, CHAACTEI!ATION, AN" H"OLSIS O$ GLCOGEN AN" $OM CHIC%EN LI&E A'uino, M() Gr(*ie+( I) "ep(rtment o Bio*-emi.tr/, $(*u+t/ o -(rm(*/ nier.it/ o S(nto Tom(.
ABSTACT Glycogen is the principal storage form of carbohydrate in the mammalian body, which is mainly present in liver liver and muscle muscles. s. This This experi experimen mentt is compo composed sed of three three parts parts:: extrac extractio tion, n, charac character teriza izatio tion, n, and and hydrolysis of glycogen. Extraction was done by heat denaturation of glycogen from chicken liver and addition of .!" acetic acid to improve precipitation. #fter extraction, $%" ethanol was used to precipitate and purify glycogen, which was seen as white precipitate. The second part involves the general tests for glycogen which includes &olisch's Test and ( ) *eaction. *eaction. +ositive results were seen on &olisch's Test and negative in ( ) *eaction which are used to test for carbohydrates and starch respectively. The glycogen ) *eaction extract produced purple interface in the &olisch's Test. or the ( ) reaction, a deep red color was expected, but there was no color change in the solution before, during and after heating. The last part of the expe experi rime ment nt is hydr hydrol olys ysis is of glyc glycog ogen en whic which h is subd subdiv ivid ided ed into into acid acidic ic and and enzy enzyma mati tic c hydr hydrol olys ysis is.. -oncentrated -l was used in acidic hydrolysis while saliva was used in enzymatic hydrolysis. The acidic and enzymatic enzymatic hydrolysates hydrolysates were sub/ected sub/ected into 0enedict' 0enedict's s Test Test and yielded yielded negative negative results, results, which which indicates that glycogen is a not a reducing sugar.
INTO"CTION Glycogen is synthesized from glucose by the pathway of glycogenesis, which occurs mainly in liver and muscle. It is the major glucose storage poly polyme merr in anim animal als. s. It has has a high highly ly-b -bra ranc nche hed d structure structure with linear chains chains connected connected by α (1-! glycosidic bonds and branched points α (1-"! every 1 in 1# gluc glucos ose e unit units. s. It allo allows ws the the imme immedi diat ate e release of glucose. $1%
Figure 1. &ranched structure of glycogen
'nzymes are proteins that act as catalysts for metabolic reactions. hey increase the rate of the reaction, but do not influence the )ind or amount of prod produc ucts ts form formed ed.. In gene genera ral, l, each each meta metabo boli lic c reaction has to be catalyzed in the living organism by its own special enzyme. $*%
Figure 2. +ydrolysis of glycogen into glucose
i)e other polysaccharides, glycogencan also unde underg rgo o hydr hydrol olys ysis is.. uri uring ng the the reac reacti tion on,, the the glucose monomer units of glycogen are separated. his is being done by the introduction of water in the glycogen molecule with the presence of strong acid or base which is summarized in igure * or it can also be due to the presence of enzymes. $/% 0mylase, an enzyme present in saliva, catalyzes the hydrolysis of the glycosidic lin)ages in starch. alivary alivary 2-amylase 2-amylase (1,-2- (1,-2--glu -glucan can glucano glucano hyd hydrola rolas se!, e!, a mono onomeri meric c calc alcium ium-bin -bind ding glycoprotein is involved in preliminary carbohydrate carbohydrate diges digestio tion. n. It cataly catalyze zes s the hydro hydrolys lysis is of intern internal al 2,13 glycosidic bonds present, yielding a mi4ture of maltose, glucose, oligosaccharides with varying lengths which constitute branched oligosac oligosacchar charides ides.. he 2-glycosi 2-glycosidic dic bond is very stable, having a spontaneous rate of hydrolysis of * 5 1 # -16 s-1 at room temperature. temperature. 2-0mylase 2-0mylase enhances this rate so enormously, that it can be cons consid ider ered ed as belo belong ngin ing g to the the most most-e -eff ffic icie ient nt 16 enzymes )nown, increasing the rate 1# -fold. $% ∼
In this e4periment, glycogen was e4tracted in chic)en liver. General tests were performed to the glycogen glycogen e4tract e4tract specific specifically ally 7olisch8 7olisch8s s est and Iodine 9eaction. Glycogen was precipitated using ethanol. or the hydrolysis, glycogen e4tract was hydrolyzed by strong acid and salivary enzyme to give an estimate of the polysaccharide content of the sample.
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METHO"OLOG I. '4traction of Glycogen from :hic)en iver 0n amount of 1/ g of chic)en liver was homogenized by using a blender. &oiling water appro4imately 6# m was poured into the homogenized chic)en liver. o precipitate the proteins, the mi4ture was heated in boiling water bath for /# minutes. o improve the precipitation, 1 m of #.1; acetic acid was added. he mi4ture was filtered and glycogen e4tract was obtained, which will be used throughout the e4periment.
:. I* 9eaction ew drops of #.#1 7 I * was added into the glycogen e4tract. he mi4ture was warmed in a water bath and cooled after. he color of the solution was observed before, during, and after heating of the mi4ture. III. +ydrolysis of Glycogen 0. 0cid +ydrolysis In a test tube, 6 m of glycogen e4tract, and 6 drops of conc. +:l was added. he mi4ture was covered with marble and boiled in a water bath for /# minutes. he acid hydrolysate was put in a refrigerator for &enedict8s est on the ne4t meeting. &. 'nzymatic +ydrolysis -ollection of 1aliva aliva was collected by rinsing the mouth with warm distilled water for a minute and the washings was put in a bea)er.
Figure 3. :hic)en liver in a blender
+reparation of 2ialyzing 0ag :ollodion solution was poured into a clean and dry hard glass (ignition! tube. >ith the tube in a horizontal position, the inside was completely coated by slowly rotating it while pouring off the e4cess collodion solution bac) into its container. he ignition tube was suspended so the inner coating of collodion solution will dry. >hen dried, the coat was loosened from inside and the membrane was slowly peeled. Figure 4. +eating of homogenized chic)en liver
II. Glycogen
In a bea)er, 1# m of glycogen e4tract and *./ m of saliva was added. he solution was stand at room temperature for /# minutes and viscosity was noted. he solution was introduced in a dialyzing bag and suspended overnight in a small flas) with 6# m distilled water. In the ne4t meeting, the solution was removed and the dialyzing bag was discarded. he solution inside the flas) was concentrated to a volume of 1# m using an open flame.
&. 7olisch8s est ew drops of 7olisch8s reagent was added into the glycogen e4tract. 0n amount of * m conc. +*= was carefully added to the side of the test tube to form a layer.
In two separate test tubes, 6 drops of acidic hydrolysate and 6 drops of enzymatic hydrolysate were added respectively. 0n amount of 1 m of &enedict8s soution was added to each hydrolysate. he mi4tures were heated in a boiling water bath at
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the same time. 0fter heating, the result was observed. ESLTS AN" "ISCSSION
dehydrates he4oses to form 6-hydro4ymethyl furfural in igure @. he furfurals further react with α-naphthol present in the test reagent as can be seen from igure A to produce a purple product which is shown in igure 1#. $"%
I. '4traction of Glycogen from :hic)en iver
Figure 7. ehydration of pentoses to form furfural
Figure 5. Glycogen e4tract
Glycogen was successfully isolated. igure 6 shows the isolated glycogen which is a yellow solution with small, white precipitate. he precipitation of the proteins was done by boiling the solution. uring heating, glycogen was left soluble in the solution while proteins were denatured and precipitated. he precipitation process was enhanced by the addition of #.1; acetic acid, he impurities or precipitate was separated from the solution by the use of gravity filtration. II. Glycogen
Figure 6. Glycogen precipitation by ethanol
Glycogen is a polymer which is used to trap the nucleic acids. In ethanol, glycogen is in soluble so it forms polymer structure which can be seen as white precipitate. $6%
Figure 8. ehydration of he4oses to form 6hydro4ymethyl furfural
Figure 9. urther reaction of furfurals with naphthol
α
-
Figure 10. ormation of purple interface
:. I* 9eaction he use of iodine is useful to distinguish starch and glycogen from other polysaccharides. Iodine yields a blue-blac) color in the presence of starch while glycogen comple4es with iodine to give a deep red color. =ther polysaccharides and monosaccharides yield no color changeB the test solution remains the characteristic brown-yellow of the reagent. Glycogen forms helical coils. Iodine atoms can then fit into the helices to form a
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glycogen-iodine comple4. tarch in the form of amylose and amylopectin has less branches than glycogen. his means that the helices of starch are longer than glycogen, therefore binding more iodine atoms. he result is that the color produced by a starch-iodine comple4 is more intense than that obtained with a glycogen-iodine comple4. $?% In the e4periment, the color of glycogen-iodine mi4ture was observed before, during, and after heating. 0s shown in igure 11, white precipitate was formed. 0fter heating, still no change in color of the mi4ture was observed. he shade of the glycogen comple4 is characteristic, that it can be recognized outwardly when the grouping of the iodine is as low as #.####* 7 at *# C:. he shading affectability reduces with e4panding temperature (ten times less delicate at 6# C:!, and upon the e4pansion of natural solvents, for e4ample, ethanol. $@% Degative results may be due to impurities or improper preparation of glycogen e4tract and iodine reagent.
Figure 10. +eating of glycogen-iodine solution
III. +ydrolysis of Glycogen 0. 0cid +ydrolysis Glycogen is a polymer of glucose. his is easily demonstrated by acid-catalyzed hydrolysis to the monosaccharide. he acid hydrolysis is addition of + EF+*= to a covalent bond. In the case of glycogen, the glycosidic covalent bonds are the target of acid hydrolysis. +eating of glycogen in t he presence of conc. +:l causes its hydrolysis into glucose because of the free aldehyde group, ma)ing glycogen a strongly reducing monosaccharide. hese glycosidic lin)ages (1- and 1-" carbons! are joining the monosaccharide in glycogen and their hydrolysis is uite random. 7any oligosaccharides form in between as intermediates eventually result as glucose. he reaction is shown asH :1*+**=11E+EF+*=--------*(:"+1*="! 0cid hydrolysis of acetals regenerates the carbonyl and alcohol components, in the case of
the glucose derivative, the result will be a tetramethyl ether of the pyranose hemiacetal. his compound will, of course, undergo typical aldehyde reactions. $A%
Figure 11. 0cid hydrolysate of glycogen
&. 'nzymatic +ydrolysis 'nzyme-catalyzed hydrolyses are more specific with respect to bonds cleaved, for e4ample, α-amylase of human saliva. he αamylase catalyzes the rapid, random hydrolysis of internal α-1, bonds. hey do not however, hydrolyze α-1," lin)ages, regardless of molecular size, nor do they hydrolyze maltose. hus, glycogen is initially split by α-amylase action into branched de4trins of medium molecular weight and only small amounts if maltose is formed. he final degradation products of the action of α-amylase on glycogen are glucose, maltose and isomaltose. he glucose is formed by the relatively slow end cleavages of the oligosaccharides. $1#% 'nzymatic hydrolysis was done by the process of dialysis, which includes a semi-permeable membrane that allows molecules to pass through via diffusion into the surrounding medium. In the e4periment, the dialyzing bag, which is a collodion solution composed of pyro4ylin film, ether, and alcohol, served as the membrane that allows monosaccharides and disaccharides to pass through into the distilled water medium. he sugar solution produced by addition of salivary enzyme into the glycogen e4tract was more viscous before hydrolysis. 0fter an hour, the solution became less viscous as shown in igure 1*.
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Figure 12. 'nzymatic hydrolysate of glycogen
0enedict's Test he &enedictJs
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