Isolation and Color Reactions of Intact Proteins

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ISOLATION AND COLOR REACTIONS OF INTACT PROTEINS

Jon Ellis G. Datu, Athina Darla B. Deala, Alan Nathan D. Derige, Mary Kimberly L. Espaldon,Ma. Theresa Angeli M. Estabillo, and Jemielle Patricia A. Estrada

ro!p " #$ Medical Technology eneral Biochemistry Laboratory

ABSTRACT%n the e&periment, the gro!p had isolated casein and alb!min 'rom non('at mil) by isoelectric precipitation and heatdenat!ration, respecti*ely. +asein as precipitated by heating the mi&t!re !p to - /+ and adding 01 acetic acid!ntil the p2 reached -.3 or !ntil a solid hite c!rd(li)e s!bstance 'ormed. 4n the other hand, the alb!min ascollected by s!b5ecting the decantate to 67 /+ ater bath 'or abo!t 7 min!tes. A'ter precipitating casein and alb!min,se*eral samples ere !sed 'or the 8!alitati*e color reactions. %ntact protein, acid hydrolysate and basic hydrolysateere !sed in per'orming Bi!ret test, Ninhydrin test, 9anthoproteic test, Millon:s test, 2op)ins(+ole test, ;a)ag!chitest, Nitropr!sside test, $ohl:s test, test 'or amides and Pa!ly test. The intact protein, casein, yielded negati*e res!lts'or Millon:s test and ;a)ag!chi test and the rest o' the tests ere positi*e. 4n the other hand, the intact protein,alb!min, obtained negati*e res!lts a'ter s!b5ecting to Ninhydrin test, 9anthoproteic test, Millon:s test, and 2op)ins(+ole test. Lastly, all test 'or amides t!rned the red litm!s paper to bl!e.

INTRODUCTION Biologically acti*e proteins are polymersconsisting o' amino acids lin)ed by co*alentpeptide bonds. Many di''erent con'ormations<three(dimensional str!ct!res= are possible 'or amolec!le as large as a protein. 4' these manystr!ct!res, one or a 'e ha*e biological acti*ity>these are called the nati*e con'ormations. Manyproteins ha*e no ob*io!s reg!lar repeatingstr!ct!re. As a conse8!ence, these proteins are're8!ently described as ha*ing large segments o' ?random str!ct!re@ also re'erred as ?randomcoil@. The term ?random@ is really a misnomer,beca!se the same nonrepeating str!ct!re is'o!nd in the nati*e con'ormation o' all molec!leso' a gi*en protein, and this con'ormation is

needed 'or its proper '!nction. Beca!se proteinsare comple&, they are de'ined in terms o' 'o!rle*els o' str!ct!re namely primary, secondary,tertiary and 8!aternary str!ct!res 0. Primarystr!ct!re is the order in hich the amino acids ina protein are lin)ed by peptide bonds 0.;econdary str!ct!re is the arrangement in spaceo' the bac)bone atoms in a polypeptide chain andit is dependent on hydrogen bonding 7. Thearrangement in space o' all the atoms in aprotein 0 and the o*erall three(dimensionalshape o' an entire protein molec!le is called thetertiary str!ct!re 7. Lastly, the 8!aternary

str!ct!re re'ers to the interaction o' se*eralpolypeptide chains in a m!ltis!b!nit protein 0. Protein isolation is a process 'or isolating asingle type o' protein 'rom a comple& mi&t!re.The signi'icance o' isolating proteins is tocharacteriCe their sol!bility, acid(base property,'!nction, str!ct!re, and interactions. Proteins canbe separated depending on their siCe, shape,charge, hydrophobicity and physiochemicalproperties. ;ome o' the methods that arecommonly !sed are isoelectric precipitation, heatdenat!ration, sol!biliCation, salt(ind!ced

precipitation, chromatography, and !ltracentri'!gation .

%n isoelectric precipitation, the isoelectric pointm!st be achie*ed herein the net charge o' the

protein ill be e8!al to Cero. %t is done byprecipitating a comple& mi&t!re !ntil the proteinis precipitated at a certain p2 le*el 6.

%n heat denat!ration, the secondary, tertiaryand 8!aternary str!ct!res o' proteins are lostand proteins are isolated based on their heatsensiti*ity. ;ome proteins denat!re at certaintemperat!res and heating ill help isolate theproteins that easily denat!re 3. ;peci'ic reactions are !sed 'or the p!rpose o'identi'ying amino acids and proteins in biologicamedia, 'or 8!alitati*e and 8!antitati*e analysis

Bi!ret test is !sed to determine peptide bondsNinhydrin test is typical 'or (amino acids9anthroproteic test is a test 'or the detection o'aromatic proteins in hich concentrated nitricacid reacts ith the proteins to 'orm a yellocolor that is intensi'ied to orange(yello by theaddition o' al)ali. Millon:s test is !sed todemonstrate the presence o' the amino acidtyrosine. 2op)ins(+ole test is speci'ic 'ortryptophan gro!p. ;a)ag!chi test is a test 'org!anidines, i.e arginine and peptides that containit. Nitropr!sside test is a test 'or cystin!ria. $ohl:stest is !sed to )no i' s!l'!r(containing amino

acids are present. Test 'or amides is !sed todetect F(gro!ps o' asparagines and gl!tamine#. %n the e&periment per'ormed, there ere toproteins that ere isolated hich are casein andalb!min. +asein as isolated thro!gh isoelectricprecipitation by acetic acid. +asein e&ists in mil)as calci!m caseinate. 4n the other hand, alb!minas isolated thro!gh heat denat!ration. Alb!minsare glob!lar proteins that are sol!ble in aterand in dil!te salt sol!tions. They are, hoe*erdenat!red and coag!lated by heat #.



The ob5ecti*es o' this e&periment are to isolatecasein and alb!min 'rom non('at mil) byisoelectric precipitation and heat denat!ration,respecti*ely and to analyCe chemical gro!psresponsible 'or color reactions and e&plain theprinciple in*ol*ed in each test.

EXPERIMENTAL

A. Compouns t!st! "o# Sampl!s us!$

Non('at mil) Mil) is a li8!id s!bstance prod!ced by themammary glands o' mammals. There are se*eraltypes o' proteins in mil) and the ma5or mil)proteins are !ni8!e to mil), it is not present inany other tiss!e. The primary gro!p o' mil)proteins are the caseins. All other proteins 'o!ndin mil) are gro!ped together !nder the name o' hey proteins. The ma5or hey proteins in comil) are beta(lactoglob!lin and alpha(lactalb!min-.B. P#o%!u#!

&. Isolation o' P#ot!insa. Cas!in '#om Non('at Mil) The gro!p placed 7. mL o' non('at mil) intoa 0 mL bea)er and heated it to abo!t - /+.Ghen the mi&t!re had reached - /+, 01acetic acid as added dropise and the sol!tionas gently stirred a'ter e*ery 7 drops !ntil thep2 reached -.3 or !ntil a solid hite c!rd(li)es!bstance 'ormed. Then, the congealed caseinas 'iltered by gra*ity 'iltration and thedecantate as reser*ed 'or the isolation o' alb!min. Lastly, the casein resid!e as alloed todry and the 1 eight o' the casein isolated 'romthe podered mil) as calc!lated.*. Al*umin '#om Non('at Mil) %n order to isolate the alb!min 'rom mil), thedecantate that as reser*ed 'rom the isolation o' casein as s!b5ected to 67 /+ heat 'or abo!t 7min!tes in a ater bath. Lastly, the li8!id 'romthe precipitated alb!min as decanted.+. ,ualitati-! Colo# R!a%tions $or each test, the gro!p prepared an intactprotein sol!tion that consists o' .7 g o' proteinin 0 mL distilled ater in separate test t!bes. The 'irst test per'ormed as the Bi!ret test

herein # drops o' #.7 M Na42 as added andmi&ed to the samples. Then, #(" drops o' .0 M+!;4- as added. The sol!tion as s!b5ected tothe *orte& mi&er and the color as noted. The second test as the Ninhydrin test herein3(0 drops o' .01 ninhydrin sol!tion as placedinto the samples then heated in a boiling aterbath. A'ter that, the appearance o' a bl!e(*ioletcoloration as noted. The third test as the 9anthoproteic testherein 0 drops o' concentrated 2N4" assloly added into the samples and the sol!tion

as mi&ed and the color as noted. A'ter adding2N4", 0 drops o' Na42 as sloly added andagain, the sol!tion as mi&ed and the color asnoted.

The 'o!rth test as the Millon:s test herein 7drops o' Millon:s reagent as added to thesamples and the change in color as obser*ed. The 'i'th test as the 2op)ins(+ole testherein # drops o' 2op)ins(+ole reagent as

sloly added to the samples. Then each test t!beas inclined and # drops o' concentrated 2#;4-

as sloly added. The color at the inter'ace asnoted. The si&th test as the ;a)ag!chi test herein0 drops o' 01 Na42 and 0 drops o' .#1naphthol sol!tion ere added to the samplesA'ter " min!tes, " drops o' #1 Na4Br as mi&edto the samples and the color prod!ced asnoted. The se*enth test as the Nitropr!sside testherein .7 mL o' " M Na42 as added to thesamples. Then, .#7 mL o' #1 nitropr!ssidesol!tion as added. Also, the color as noted.

The eighth test as the $ohl:s test herein 7drops o' "1 Na42 and # drops o' 71<+2"+44=# Pb as added to the samples then thet!bes ere placed in a boiling ater bath and theappearance o' dar) <bron or blac)= sedimentas noted don.

The ninth test per'ormed as the test 'oramides herein 0 mL o' #1 Na42 as added to0 drops o' the sample. The samples ere thenplaced in a boiling ater bath and red litm!spaper as placed and moistened o*er the mo!th

o' the t!be. The last test per'ormed as the Pa!ly testherein the gro!p prepared the diaCo reagent bymi&ing "(7 drops 01 s!l'anilic acid ith " drops71 NaN4# sol!tion. Then, 7 drops o' the sampleand "(7 drops o' 01 Na#+4" ere added to theprepared diaCo reagent and the appearance o'red coloration as noted.

RESULTS AND DISCUSSION

A. Isolation o' Cas!in '#om Non('at Mil) +asein is separated 'rom mil) by acidi'icationto bring it to its isoelectric point. Ghen 01acetic acid as added to the non('at mil) mi&t!reat a controlled p2 le*el, yelloish hiteprecipitate as prod!ced. That p2 *al!e is )nonas the isoelectric point o' the protein and isgenerally the p2 at hich the protein is leastsol!ble. $or casein, the isoelectric point isappro&imately -.3 and it is the p2 *al!e at hichacid casein is precipitated. This process is calledisoelectric precipitation. Precipitation occ!rredbeca!se the protein has already reached itsisoelectric point herein its net charge is Cero6.



Fiu#! &. %solation o' +asein 'rom Mil)

Fiu#! +. +asein 'rom Mil)

B. Isolation o' Al*umin '#om Non('at

Mil) A'ter the casein as separated, alb!min as

e&tracted ne&t !sing heat denat!ration. To beable to get the alb!min, the e&tract 'rom thecasein as heated. This o!ld ca!se destr!ctionto the tertiary le*el o' the s!bstance enabling thegro!p to get the alb!min. As shon on 'ig!re "the color o' Alb!min is slightly yello.

Fiu#! /. %solation o' Alb!min 'rom Mil)

C. ,ualitati-! Colo# R!a%tion %ntact proteins alb!min and casein ere !sedto determine hich proteins ill gi*e a positi*e ornegati*e res!lt to a speci'ic reaction test.

Ta*l! &. H!alitati*e +olor Feaction o' %ntacProteins

Colo# R!a%tionInta%t P#ot!in

"Cas!in$

Inta%t P#ot!in

"Al*umin=

Biu#!t T!st Iiolet IioletNin01#in T!st Bl!e(*iolet +olorlessXant0op#ot!i%

T!st

acid ello base4range

acid+olorless base+olorless

Millon2s T!st +olorless +olorless3op)ins(Col!

T!st

Iiolet ring +olorless

Sa)au%0i T!st +olorless Ghite(t!rbidNit#op#ussi!

T!st

ello ello

Fo0l2s T!st Bron

sediment

Bron

sedimentT!st 'o# Ami! Fed(Bl!e

litm!s paperFed(Bl!elitm!s paper

Paul1 T!st Fed(orange ello(orange

The Bi!ret Test is a general test done toproteins to detect peptide lin)ages. Positi*eres!lts sho the 'ormation o' *iolet sol!tion. Theprinciple in*ol*ed in this test is comple&ationreaction hich ta)es place beteen a metal ionand a molec!lar or ionic entity )non as a ligand. ;ince the test res!lts positi*e 'or peptide



lin)ages, as seen in the res!lts, only the intactproteins o' alb!min and casein res!lted positi*e. Ninhydrin Test is !sed to detect the 'ree alphaamino gro!ps. The !nderlying principle behindthis test is o&idati*e deamination anddecarbo&ylation. A positi*e res!lt 'or the testo!ld sho a bl!e(*iolet coloration o' thesol!tion . Based on the res!lts, only the intactcasein res!lted positi*e. The intact alb!min

res!lted negati*e indicating that 'ree alpha aminogro!ps are not present ithin the protein. 9anthoproteic test indicates the presence o' acti*e aromatic gro!ps that are deri*ati*es o' benCene in amino acids s!ch as tyrosine andtryptophan. The principle in*ol*ed in the test isthe nitration o' the acti*ated benCene ringthro!gh nitric acid. The nitration res!lts to ayello to orange sol!tion . Based on theres!lts, the intact protein casein has a positi*eres!lt. Millon:s test is !sed to detect the presence o' tyrosine. The principle in*ol*ed in the test is thecomple&ation reaction beteen the phenolicgro!ps and merc!ry present in the Millon:sreagent. Positi*e res!lt shos the salmon pin)coloration o' the sol!tion . Both intact caseinand alb!min shoed a negati*e res!lt. 2op)ins(+ole Test is !sed to detect thepresence o' tryptophan. %t is g!ided by theprinciple o' condensation o' the indole gro!p ithglyo&ylic acid and 2#;4-. A positi*e res!lt o' thetest shos the 'ormation o' a *iolet ring or thecoloration o' to layers. %n other cases, a similarres!lt is prod!ced hen s!l'!ric acid is added to

the sol!tion in presence o' 'ormaldehyde ".4nly the intact casein prod!ced a positi*e res!lt. ;a)ag!chi test is !sed to detect arginine, theonly amino acid that contains a g!anido gro!p.The principle behind the test is that the g!anidogro!p reacts ith naphthol, in the presence o' ano&idiCing agent, s!ch as sodi!m hypobromite.The reaction prod!ces a red or orange colorationo' the sol!tion . %t is negati*e to both caseinand alb!min beca!se they prod!ce a colorlessand hite(t!rbid sol!tion, respecti*ely. Nitropr!sside test is !sed to determine thepresence o' cysteine, the only amino acid that

contains the s!l'hydryl gro!p. The principlebehind this test is comple&ation> the s!l'hydrylgro!p reacts ith nitropr!sside in presence o' e&cess ammonia. +ysteine is partially destroyedand a positi*e res!lt shos a red or yellocomple& ". Both casein and alb!min shoedpositi*e res!lts. $ohl:s test determines the presence o' s!l'!r(containing proteins s!ch as methionine andcysteine. The test is g!ided by the principle o' '!sion 'olloed by ionic interaction. A positi*eindicator 'or the test is the 'ormation o' blac) or

bron precipitate 'rom lead s!l'ide ". A positi*eres!lt as obser*ed 'or both intact proteins. Test 'or amides is !sed to determine the F(gro!ps o' asparagine and gl!tamine. A positi*eres!lt shos the change o' red litm!s paper tobl!e litm!s paper ". +asein and alb!minres!lted positi*e 'or the test. Pa!ly:s test detects the presence o' tyrosine orhistidine hich deals ith the 'ormation o' aCo

dyes. Ghen proteins containing either tyrosine orhistidine are reacted ith diaCotiCed s!l'anilicacid, a red to yello coloration is 'ormed . Theintact proteins o' casein prod!ced the strongestpositi*e res!lt.

Fiu#! 4. +olor Feaction o' +asein

Fiu#! 5. +olor Feaction o' Alb!min

REFERENCES0 +ampbell, M.K., and $arrell, ;.4. <#07=Biochemistry. th ed. Lorong +h!an, ;ingapore+engage Learning# +risostomo, A.+., Daya, M.L., de !ia, F.M.,$arro, $.L., abona, M.., Li!, M.%.D., Pena.T., Pena, L.L., ;antiago, L.A., ;antiago, M.F.;arile, A.;., Torres, P.+., Iargas, A.., and srael

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Isolation and Color Reactions of Intact Proteins

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