qualitative color reactions of albumin

7/26/2019 Qualitative Color Reactions of Albumin

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QUALITATIVE COLOR REACTIONS OF INTACT PROTEIN AND ITS HYDROLYSATES(ALBUMIN)

Zoe Angela M. Espinosa, Ma. Pamela Rica M. Fajardo, Emmanuel Joshua M. Garcia,Liana Celina D. Gelu, Jillian Pamela D. !, and Jenni"e# Mae C. He#e$ia

Group ! "A# Medical $echnolog% C&EM '(( La)ora*or%

ABSTRACT$he +uali*a*ie color reac*ions o- al)umin ere *es*ed *hrough color reac*ions con-irming *he presence o- cer*ainamino acids in )o*h *he in*ac* al)umin and in i*s h%drol%sa*es. $he /iure* *es* con-irmed *he presence o- pro*ein in allsamples i*h each giing a )lue#iole* colora*ion, hile *he 0inh%drin *es* de*ec*ed *he presence o- amino acidscomple*el% h%drol%ed in *he )asic h%drol%sa*e, as i* as *he onl% sample hich gae a )lue#iole* solu*ion. $he1an*hopro*eic *es* de*ec*ed *he presence o- an aroma*ic amino 2*%rosine or *r%p*ophan3 as posi*ie resul*s 2%ellosolu*ion3 ere o)sered in al)umin. $his aroma*ic amino acid as speci-ied *hrough *he Millon *es* and *hrough *hePaul% *es* as all samples *es*ed nega*ie -or )o*h reac*ions speci-ic -or *%rosine. $he presence o- *r%p*ophan as *hencon-irmed *hrough *he &op4ins#Cole *es* hich gae a red ring a* *he junc*ion ins*ead o- a purple ring. 5n *a4ing no*eo- *he errors commi**ed in *he e6perimen*, *his as alread% considered a posi*ie resul*. $he 7a4aguchi *es* and *he*he *es* -or amide )o*h con-irmed *he presence o- arginine as a non#*erminal amino acid, in giing a nega*ie resul*-or *he in*ac* pro*ein )u* a posi*ie resul* -or a* leas* one o- *he h%drol%sa*es. $he Fohl8s *es* de*ec*ed *he presence o-sul-ur#con*aining samples 2ei*her c%s*eine or me*hionine3 as a dar4 )ron solu*ion as o)sered -rom *he resul*s$his amino acid as -ur*her speci-ied as me*hionine i*h *he nega*ie resul* o- *he )asic h%drol%sa*e in Fohl8s *es* andi*h *he nega*ie resul*s o- *he 0i*roprusside *es* speci-ic -or c%s*eine. $he color *es* reac*ions ere a)le *o de*ec* *hepresence o- *r%p*ophan, arginine, and me*hionine in al)umin.

INTRODUCTIONAl)umin is a a*er#solu)le, hea* coagula)le

pro*ein. Al)umin is presen* in egg hi*e 2eggal)umin3, mil4 2lac*al)umin3 and in serum. Egg

al)umin and lac*al)umin are gl%coconjuga*edpro*eins, hereas serum al)umin is a simple

pro*ein. Al)umin is a pro*ein o- high )iologicalalue i*h all essen*ial amino acids 9:(;.

Amino acids hae a arie*% o- chemicall%

reac*ie groups. $he reac*ions -or side chains, #

amino and #car)o6%l groups can )e used *o

charac*erie )o*h -ree amino acids and pro*eins9";. 5n charac*eriing *he amino acids presen* in

al)umin, *he principle )ehind +uali*a*ie colorreac*ions is emplo%ed *hrough di--eren* *es*s and

reagen*s. /iure* *es* is used *o de*ec* *he presence o- a

pro*ein or pep*ides. 5*s posi*ie resul* is a iole*

colora*ion hich means *ha* i* is a su)s*ance2pro*ein3 *ha* con*ains no* less *han *o pep*ide

lin4ages 9s reagen**o de*ec* phenolic

compounds such as *%rosine. 5*s posi*ie resul* is

a red precipi*a*e or a red solu*ion 9=;.&op4ins#Cole *es* is a speci-ic -or *r%p*ophan

5*s posi*ie resul* is a pin4 *o purple in*er-ace 9


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(.= mL each o- *he h%drol%ed samples 2acid,)asic, en%ma*ic3 as used.

+. Bi'#e Te $en*% drops o- ".= M 0a?& as in*roduced

*o *he samples and mi6ed ell. $hen, "#@ dropso- (.: M Cu7?! solu*ion as added *o *he

solu*ions. $he *es* *u)es ere *hen sha4en *omi6 *he solu*ion ell, and *he colors ere no*ed

don.

,. Nin-$#in Te 7i6 *o *en drops o- (.: ninh%drin solu*ionas placed in*o dilu*ed samples. $he *u)e as

*hen hea*ed in a )oiling a*er )a*hB a )lue#iole*colora*ion as no*ed don.

/. %an-!!ei* Te

$en drops o- concen*ra*ed &0?@as slol%added *o *he dilu*ed samples. 5* as mi6ed, and

*he color o- *he solu*ion as no*ed. $en drops o-

concen*ra*ed 0a?& as slol% added. 5* as alsomi6ed, and i*s color as no*ed.

0. Mill!n1 Te Fie drops o- Millon8s reagen* as added *o

*he dilu*ed samples. $he change in color asno*ed.

2. H!&3in4C!le Te $en*% drops o- &op4in8s#Cole reagen* as

slol% added *o *he samples. 5* as *hen mi6edell. $he *es* *u)e as inclined, and along i*s

side, *en*% drops o- concen*ra*ed &"7?! assslol% added. $he color a* i*s in*er-ace as

no*ed.5. Sa3a6'*-i Te

$en drops o- :( 0a?& as added. $en dropo- (." naph*hol solu*ion as also added *o *he

samples. 5* as *hen mi6ed and le* s*and -or*hree minu*es. $hree drops o- " 0a?/r as

added. 5* as *hen mi6ed, and *he colorproduced as no*ed.

7. Ni#!'i$e TeA hal- millili*er o- @ M 0a?& as added *o *he

(.= mL o- *he sample. A +uar*er milllili*er o- "

ni*roprusside solu%*ion as *hen added. $he-orma*ion o- a red solu*ion as no*ed.

8. F!-l1 Te Fie drops o- @( 0a?& as addedB *o

drops o- = 2C&@C??3"P) as *hen added *o *he

samples. $he *u)e as placed in a )oiling a*er

)a*h. A-*er *ha*, *he appearance o- dar4 2)lac4 or)ron3 sedimen* as no*ed.

9. Te "!# Ami$e

?ne millili*er o- "( 0a?& as added *o *endrops o- *he sample. $he *u)e as placed in a

)oiling a*er )a*h. $he eolu*ion o- gas duringhea*ing as *es*ed )% placing a mois*ened red

li*mus paper oer *he mou*h o- *he *u)e.+:.Pa'l Te

$he diao reagen* as prepared )% mi6ing @#= drops o- : sul-anilic acid i*h @ drops o- =

0a0?"solu*ion. Fie drops o- *he sample and @#=

drops o- :( 0a"C?@ as added *o *he diaoreagen*. $he appearance o- a red colora*ion as

no*ed.

RESULTS AND DISCUSSIONPro*eins, due *o *he presence o- pep*ide )onds

and di--eren* amino acid residues, reac* i*h a

arie*% o- reagen*s *o -orm colored produc*s$hese *es*s are 4non as color reac*ions o

pro*eins. 7eeral o- *hese reac*ions are impor*an*in +uali*a*ie de*ec*ion and +uan*i*a*ie

es*ima*ion o- pro*eins and amino acids 9:(;.$he samples used -or *he +uali*a*ie color

reac*ions include *he in*ac* pro*ein, acidich%drol%sa*e, )asic h%drol%sa*e, and en%ma*ic

h%drol%sa*e o- *he isola*ed pro*ein al)umin -oundin s4immed mil4. $he samples ere all *es*ed *o

charac*erie and de*ermine *he -unc*ional groups*ha* *he% con*ain, depending on *heir reac*ions *o

*he reagen*s o- each *es*. /iure* *es* is a general *es* -or pro*eins and is

a *es* -or de*ec*ing pep*ide lin4age. $he )iure*

reagen* 2copper sul-a*e in a s*rong )ase3 reac*si*h pep*ide )onds in pro*eins *o -orm a )lue *o

iole* comple6 4non as *he /iure* comple6. A*

leas* *o pep*ide )onds are re+uired -or *he-orma*ion o- *his comple6 9:;.

Fi6'#e +. Pro*ein#copper2553 ion comple6, also

called *he )iure* comple6 /ased on *he resul*s o)*ained, *he in*ac*

pro*ein shoed a posi*ie resul* 2purple solu*ion3-or *his *es* since i*s pep*ide lin4age is no* %e*

)ro4en. $his goes *he same -or *he acidic anden%ma*ic h%drol%ed samples hich also

presen*ed a posi*ie resul* 2purple solu*ion3 -or*he )iure* *es*. $his means *ha* *he acidic and

en%ma*ic h%drol%sis eren8* a)le *o comple*el%h%drol%e *he sample unli4e in *he )asic

h%drol%sis hich presen*ed a clear )lue solu*iondepic*ing a loer in*ensi*% o- color correla*ing *o

*he presence o- h%drol%ed pro*ein in *he -orm o-

amino acids.Ta;le +.Resul*s o- *he /iure* $es*

Bi'#e Te


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5n*ac* Pro*ein Purple solu*ion

Pro*ein

&%drol%sa*e

Acidic Purple solu*ion

/asic Clear ligh* )lue solu*ion

En%ma*ic Purple solu*ion

0inh%drin is a *es* -or de*ec*ing -ree #amino

groups. $he coalen*l% )onded pep*ide )onds

presen* in pro*eins are disrup*ed as *he% areh%drol%ed. A-*er comple*ion o- h%drol%sis, *he

h%drol%ed pro*ein or h%drol%sa*e pro*ein is ha*

remains, con*aining a mi6*ure o- amino acids *ha*reac* i*h ninh%drin. 0inh%drin reac*s i*h #

amino acids 2#0&"3 in pro*eins *o gie a purple

comple6 as illus*ra*ed in *he reac*ion *ha* -ollos,i*h *he e6cep*ion o- proline and h%dro6% proline

hich )o*h gie a %ello color due *o *he a)sence

o- -ree #amino acid. 0inh%drin is mos*

commonl% used as a -orensic chemical *o de*ec*

-ingerprin*s as amines le-* oer -rom pro*eins,sloughed o-- in -ingerprin*s, reac* i*h ninh%drin

*o gie a charac*eris*ic purple color 9:;.

Fi6'#e ,. 0inh%drin reac*ion -orming a purplecomple6

$he principle )ehind *his *es* is o6ida*iedeamina*ion i*h a -ree amino group, #0&". $he

amino group is released as ammonia 20&@3. Aposi*ie indica*ion o- *his *es* ould )e a )lue#

iole* colora*ion in *he solu*ion, hich deepens as*he num)er o- amino group increases. 5n *he

resul*s o)*ained, onl% *he )asic h%drol%sa*e*es*ed posi*ie -or *his *es* as *he in*ac* pro*ein,

acidic h%drol%sa*e, and en%ma*ic h%drol%sa*e allpresen*ed a colorless solu*ion in *he presence o-

ninh%drin. 5* can )e said *ha* *he )asich%drol%sa*e is *he onl% sample hich produced

*he mos* -ree #amino groups as a resul* o- i*s

comple*e h%drol%sis.

Ta;le ,. Resul*s o- *he 0inh%drin $es*Nin-$#in Te

5n*ac*

Pro*einColorless solu*ion

Pro*ein

&%drol%sa*e

Acidic Colorless solu*ion

/asic /lue#iole* solu*ion

En%ma*ic Clear colorless solu*ion 1an*hopro*eic *es* is a *es* -or *he presence o-

aroma*ic amino acids hich include *%rosine and*r%p*ophan, i*h phen%lalanine no* readil%

ni*ra*ed and so does no* respond *o *his *es* as i*is ordinaril% per-ormed. Aroma*ic amino acids

reac* i*h concen*ra*ed &0?@ a* high

*empera*ures *o -orm ni*ro#compounds hich are%ello in color. A* al4aline p&, *he color changes

*o orange due *o *he ionia*ion o- *he phenolic

group 9;.

Fi6'#e /. Aroma*ic amino acid reac*ion i*hconcen*ra*ed &0?@-orming a ni*ro#compound

5n *he resul*s o)*ained, onl% *he in*ac* pro*ein

and *he acidic h%drol%sa*e %ielded a posi*ie

resul* 2clear %ello solu*ion3 hile *he )asic anden%ma*ic h%drol%sa*es )o*h resul*ed *o a clearcolorless solu*ion. From *his, i* can )e said *ha*

an aroma*ic amino acid 2*%rosine or *r%p*ophan3is presen* on *he *erminal side o- *he pro*ein

al)umin in %ielding a posi*ie resul* -or *he in*ac*pro*ein. 5* can also )e said *ha* *his aroma*ic

amino acid as procured during *he acidich%drol%sis o- *he in*ac* pro*ein, hile i* is

possi)le *ha* *he amino acid as des*ro%edduring )asic and en%ma*ic h%drol%sis.

Ta;le /.Resul*s o- *he 1an*hopro*eic $es*%an-!!ei* Te

5n*ac*

Pro*ein

# Clear hi*e solu*ion

# Clear %ello solu*ion i*

cloud% -orma*ion

Pro*ein

&%drol%sa*e

Acidic

# Clear hi*e solu*ion

# Clear %ello solu*ion

/asic Mil4% hi*e solu*ion

En%ma*ic Clear colorless solu*ion

Millon8s *es* is a *es* speci-ic -or *%rosine, *heonl% amino acid con*aining a phenol group, a

h%dro6%l group a**ached *o a )enene ring. 5n

Millon8s *es*, *he phenol group o- *%rosine is -irs*

ni*ra*ed )% ni*ric acid in *he *es* solu*ion. $hen*he ni*ra*ed *%rosine comple6es mercur%253 andmercur%2553 ions in *he solu*ion *o -orm ei*her a

red precipi*a*e or a red solu*ion, )o*h posi*ieresul*s 9@;.

Fi6'#e 0.Millon Reac*ion Principle ?)sera*ions on *he resul*s o)*ained shoed*ha* nei*her o- *he in*ac* pro*ein nor an% o- *he

pro*ein h%drol%sa*es *es*ed posi*ie -or *his *es*,i*h all resul*s giing a clear hi*e or cloud%

solu*ion.i*h *his resul*, *%rosine can )e elimina*ed

-rom *he lis* o- aroma*ic amino acids presen* in

al)umin, leaing *r%p*ophan as *he aroma*icamino acid procured during *he acidic h%drol%sis

o- *he in*ac* pro*ein during *he preiousl% done

1an*hopro*eic *es*.


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Ta;le 0. Resul*s o- *he Millon8s $es*Mill!n1 Te

5n*ac* Pro*ein Clear hi*e

Pro*ein&%drol%sa*

e

Acidic Colorless clear solu*ion

/asic Cloud% hi*e solu*ion

En%ma*ic Clear hi*e solu*ion

$he &op4ins#Cole *es* is speci-ic -or*r%p*ophan, *he onl% amino acid con*aining an

indole group. As *he pro*ein solu*ion is

h%drol%ed )% *he concen*ra*ed sul-uric acid a**he solu*ion in*er-ace, *he indole ring presen* in*r%p*ophan reac*s i*h gl%o6%lic acid *o -orm a

purple ring produc* 9@;. Concen*ra*ed sul-uricacid and insolu)le a*er ill no* mi6 a* once

hich ill resul* *o *he -orma*ion o- *o la%ersi*h a purple ring on *he sur-ace o- *he solu*ion

junc*ion.

Fi6'#e 2.Purple ring -orma*ion -rom *he reac*ion

o- an indole ring in *r%p*ophan i*h gl%o6%lic acid$he in*ac* pro*ein in *his e6perimen* as

o)sered *o -orm a red ring a* *he junc*ion here

i* should hae resul*ed *o a purple ring. Posi*ieresul*s are s*ill recorded -or *he in*ac* pro*ein,

i*h a no*e indica*ing some errors in *he

e6perimen* *ha* ma% hae des*ro%ed or lessened*he amino acid presen* in *he sample. $he pro*ein

h%drol%sa*es, on *he o*her hand, presen*ednega*ie resul*s, shoing *o la%ers o- a clear

and cloud% solu*ion. $his means *ha* *r%p*ophanis presen* a* *he *erminal side o- *he in*ac*

pro*ein hich ma% hae )een des*ro%ed orlessened during h%drol%sis.

$his resul* con-irms *he preiousl% concludedpresence o- *r%p*ophan on *he *erminal side o-

al)uminB *he presence o- an aroma*ic amino acidde*ec*ed )% *he 1an*hopro*eic *es* and *he amino

acid speci-ied )% )o*h *he Millon *es* and *he&op4ins#Cole *es*.

Ta;le 2.Resul*s o- *he &op4ins#Cole $es*H!&3in4C!le Te

5n*ac*

Pro*ein Red ring a* *he junc*ion

Pro*ein

&%drol%sa*

e

Acidic Clear colorless solu*ion

/asic

" la%ers 2mil4% hi*e a*

junc*ion3

# Cloud% hi*e solu*ion

# Clear colorless solu*ion

i*h )u))les

En%ma*ic Clear colorless solu*ion

7a4aguchi *es* is a *es* -or *he amino acid

con*aining *he guanidine group in Arginine 9:;.$he guanido 20&"#C0&3 o- -ree arginine or

arginine residues in *he pro*ein reac*s i*h #

0aph*hol and sodium h%po)romi*e *o gie a

)righ* red#orange colored comple6 9:(;.

Fi6'#e 5. Comple6 -orma*ion -rom guanidine

group reac*ion i*h #0aph*hol and sodiumh%p)romi*e.

7ince al4aline or )asic h%drol%sis des*ro%s

arginine, *he )asic h%drol%sa*e presen*ed anega*ie resul* 2clear ligh* )ron solu*ion3 hile

*he acidic h%drol%sa*e presen*ed a posi*ie resul*2orange solu*ion3. $he nega*ie resul* o)*ained

-rom *he reac*ion i*h *he in*ac* pro*ein2colorless solu*ion i*h a cloud% -orma*ion3

indica*es *ha* *he arginine is a non#*erminaamino acid hile *he en%ma*ic h%drol%sa*e

hich had a clear ligh* )ron solu*ion same *o*ha* o- *he )asic h%drol%sa*e indica*es *ha

arginine as also des*ro%ed during en%ma*ich%drol%sis.

Ta;le 5.Resul*s o- *he 7a4aguchi $es*Sa3a6'*-i Te

5n*ac*

Pro*ein

Colorless solu*ion i*h

cloud% -orma*ion

Pro*ein

&%drol%sa*

e

Acidic ?range solu*ion

/asic Clearl ligh* )ron solu*ion

En%ma*icHer% clear ligh* )ron

solu*ion

0i*roprusside *es* is speci-ic -or c%s*eine, *heamino acid con*aining a sul-h%dr%l group 2#7&3

$he group reac*s i*h ni*roprusside in al4alinesolu*ion *o %ield a red comple6 9@;. $he -orma*ion

o- *his red#purple colored comple6 is also called*he MIrner *es* 9:;.

Fi6'#e 7. Red comple6 -orma*ion -rom *he

reac*ion o- ni*roprusside i*h c%s*eine.?)sera*ions on *he resul*s o)*ained shoed

*ha* nei*her o- *he in*ac* pro*ein nor an% o- *he

pro*ein h%drol%sa*es *es*ed posi*ie -or *his *es*,i*h all resul*s giing a clear %ello or dar4%ello#orange solu*ion.

Ta;le 7.Resul*s o- *he 0i*roprusside $es*Ni#!'i$e Te

5n*ac* Pro*ein Clear %ello solu*ion

Pro*ein

&%drol%sa*

e

AcidicDar4 %ello#orange

solu*ion

/asic Clear %ello solu*ion

En%ma*ic Clear %ello solu*ion

Fohl8s *es* is a *es* -or sul-ur#con*aining

pro*eins. 5* also indica*es *he presence o


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me*hionine and c%s*eine hich are amino acids)o*h con*aining sul-ur in *heir s*ruc*ures. A

posi*ie resul* -or *his *es* is *he -orma*ion o-)lac4 precipi*a*e -rom lead sul-ide 2P)73. $he

dar4 colora*ion o- *he in*ac* pro*ein and *heacidic h%drol%sa*e indica*es *he presence o- sul-ur

-rom *hese samples hile *he )asic h%drol%sa*epresen*ed a nega*ie resul* hich ma% indica*e

*ha* *he amino acid presen* is me*hionine, as i*s

sul-ur is no* readil% remoed )% al4ali and is no*a--ec*ed )% *his reac*ion.

Ta;le 8.Resul*s o- *he Fohl8s $es*F!-l1 Te

5n*ac*

Pro*einDar4 )ron in color solu*ion

Pro*ein

&%drol%sa*e

Acidic Dar4 )ron solu*ion

/asic Clear colorless solu*ion

En%ma*i

cClear ligh* %ello solu*ion

$he *es* -or amides indica*es *he presence o-

primar%, secondar% and *er*iar% amides 2in

asparagine and glu*amine3 and ni*riles 2inarginine3. A posi*ie resul* -or *his *es* is *he

change in color o- li*mus paper -rom red *o )lue,as a resul* o- *he process o- )asic h%drol%sis.

/asic h%drol%sis causes par*ial or comple*edecomposi*ion o- c%s*eine, arginine, and l%sine.

$he amide groups o- glu*amine and asparaginerelease ammonia and *he sul-ur#con*aining amino

acid, c%s*eine, is par*iall% des*ro%ed *o releaseuno6idied sul-ur in *he h%drol%sa*e 9';.

All *he pro*ein h%drol%sa*es gae a posi*ieresul* -or *his *es*, *urning *he li*mus paper -rom

red *o )lue hile *he in*ac* pro*ein as o)sered*o gie a neu*ral resul* i*h *he red li*mus paper

unchanged. $his con-irms *he presence o-arginine 2-rom 7a4aguchi *es*3 )eing a non#

*erminal amino acid presen* in al)umin as *hein*ac* pro*ein presen*ed nega*ie resul*s.

Ta;le 9.Resul*s -or *he $es* -or AmideTe "!# Ami$e

5n*ac* Pro*ein Red red li*mus paper

Pro*ein

&%drol%sa*

e

Acidic Red )lue li*mus paper

/asic Red )lue li*mus paper

En%ma*icClear ligh* %ello solu*ion

2red )lue li*mus paper3

Paul% $es* is speci-ic -or *he de*ec*ion o-*%rosine and his*idine. $he reagen* used -or *his

*es* con*ains sul-anilic acid dissoled inh%drochloric acid. 7ul-anilic acid upon

diao*ia*ion in *he presence o- sodium ni*ri*eand h%drochloric acid resul*s in *he -orma*ion o- a

diaonium sal*. $he diaonium sal* -ormedcouples i*h *he imidaole ring o- his*idine or *he

phenol group o- *%rosine in al4aline medium *ogie a red#colored chromogen 2ao d%e3 9;.

$he reac*ion o- *he in*ac* pro*ein and *hepro*ein h%drol%sa*es o)sered using *his *es*

shoed nega*ie resul*s, all samples giing a%ello#orange *o orange solu*ion. $his -ur*her

con-irms *ha* *%rosine is no* presen* in al)uminas as alread% o)sered -rom *he nega*ie

resul*s o- *he Millon *es*.Ta;le +:.Resul*s o- *he Paul% $es*

Pa'l Te

5n*ac* Pro*ein ello orange solu*ion

Pro*ein&%drol%sa*

e

Acidic ello orange solu*ion

/asicClear ligh* orangesolu*ion i*h hi*e

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qualitative color reactions of albumin

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