Isolation, Acid Hydrolysis & Color Reactions of Hydrolyzed Myoglobin

ISOLATION, ACID HYDROLYSIS & COLOR REACTIONS OF HYDROLYZED MYOGLOBIN Zarah G. Agbuya, Agbuya , Janmaverick D A!arci", A!arci", Adrian R. Alvarez, Trisha V. Aquino, Daniel O. Aracan, and Elain I. Arenas Grou ! "# $har%acy #iological &he%is'ry (abora'ory (abora'ory The e)eri%en' involved 'he isola'ion o* %yoglobin, hydrolysis o* 'he in'ac' ro'ein and 'he quali'a'ive reac'ions o*  hydrolyzed %yoglobin. +yoglobin as isola'ed *ro% bee*. Acid hydrolysis dena'ured 'he in'ac' ro'ein. Di**eren' quali'a'ive reac'ions ere e%loyed 'o 'es' 'he hydrolyzed %yoglobin. Only -inhydrin 'es', an'horo'eic 'es' and -i'rorusside 'es' gave nega'ive resul's. #iure' reac'ion accoun'ed *or de'er%ina'ion o* 'he resence o* e'ide bonds, -inhydrin reac'ion as e%loyed *or /0a%ino acids. an'hroro'eic 'es' as used 'o de'ec' 'he side0chains o*  aro%a'ic a%ino acids. +illon1s 'es' as e%loyed *or 'he de'er%ina'ion o* 'yrosine residues. 2o3ins0&ole 'es's as e%l e%loy oyed ed *or *or 'he 'he de' de'er er%i %ina' na'io ion n o* 'ry 'ry'o 'oh han an res resid idue ues. s. 4a3 agu chi reac 'io n as use d 'o de' ec' arg ini ne. -i'rorusside 'es' as used 'o 'es' *or sul*ur con'aining a%ino acids. 5ohl1s 'es' as also used 'o 3no i* sul*ur0 con'aining a%ino acids ere resen'. Tes' *or a%ides de'er%ined 'he resence o* a%ides. $auly Tes' de'ec'ed 'he resence o* ni'rous acid and hydrochloric hydrochloric acid.

INTROD#CTION $ro'eins, along i'h liids and carbohydra'es, are i%or'an' i%or'an' classes o* bioche%ica bioche%icall %olecules %olecules hich are essen'ial *or li*e. $ro'eins serve as 'he basis basis *or 'he %a6or %a6or s'ruc' s'ruc'ura urall co%on co%onen' en's s o*  'issues co%rising a living organis% seci*ically hu%ans and ani%als. $ro'eins $ro'eins are 'he na'ural na'ural oly%ers oly%ers o* a%ino a%ino acids. A%ino acids are covalen'ly lin3ed 'hrough e'id e'ide e bonds bonds 'o *or% *or% linear linear oly%e oly%ers rs calle called d e'ides or olye'ide chain. 7!8 The hysical and and che%ic che%ical al roer roer'ie 'ies s hich hich are unique unique 'o each a%ino acid are 'he resul' o* 'he s'ruc'ure and che%ica che%icall roer'ies roer'ies o* 'he R grou. grou. A%ino A%ino acids acids have have di**er di**eren' en' che%ic che%ical al reac'i reac'ive ve grous grous roviding a ide range o* reac'ivi'y ro'eins. The reac'ions *or individual side0chain radicals and /0 a%ino a%ino and /0carbo) /0carbo)yl yl grous grous are seci*ic seci*ic bo'h *or *or *ree *ree a%in a%ino o acid acids s and and ro' ro'ei eins ns.. 798 798 4o 4o%e %e reac'ions reac'ions used *or iden'i*ying iden'i*ying a%ino a%ino acids acids and ro'eins in biological %edia, *or quali'a'ive and quan'i'a' quan'i'a'ive ive analysis analysis include include #iure', #iure', -inhydrin, -inhydrin, an'hroro'eic, +illon, 2o3ins0&ole, 4a3aguchi, -i'rorus -i'rorusside, side, 5ohl, Tes' Tes' *or A%ides A%ides and $auly $auly 'es's. +yoglobin +yoglobin is a 3ind o* ro'ein ro'ein *ound in 'he %usc %uscle le 'iss 'issue ue o* ver' ver'eb ebra ra'e 'es. s. I' is a single0 chain globular chain  globular ro'ein o* !:; or !:9 a%ino acids, con'aining a he%e he%e,, hich is an iron iron0con'aining 0con'aining orhyrin ros'he'ic grou  grou  in 'he cen'er around hic hich h 'he 'he re%a re%ain inin ing g ao aoro ro'e 'ein in *old *olds. s. 7.;: >.;: g o* @-2 @-29"4O9 crys'als ere grounded 'o *ine oder and %i)ed gen'ly. The sa%le as cen'ri*uged again *or : %inu'es. The The sue suern rna' a'an an'' as as deca decan' n'ed ed so 'ha' 'ha' 'he 'he sa%le %ay be hydrolyzed. 7;8 3 Aci( H0(r"!0)i) ". In*ac* %r"*ein 5or 'his 'ye o* hydrolysis, !> %l o* 'he %inu'es i'h 'e%era'ures u 'o ">>o&. This ill vaorize 'he dros o* ".:+ -aO2 as added 'o one 'es' 'ube and %i)ed. ; Dros o* &u4O9 solu'ion as also added. The 'es' as sha3en and as no'ed *or color changes. 7;8 6 5'a!i*a*ive C"!"r Reac*i"n)/ Nin70(rin Te)* In -inhydrin 'es', dros o* >.!? -inhydrin solu'ion as laced in'o 'he dilu'ed sa%les and hea'ed in a a'er ba'h. The aearance o* a blue0viole' colora'ion as no'ed. 7;8 8 5'a!i*a*ive C"!"r Reac*i"n)/ $an*7"r"*eic Te)* In an'hroro'eic 'es', !> dros o* concen'ra'ed 2-O;  and !> dros o* concen'ra'ed -aO2 ere added sloly and 'hen %i)ed. &olor changes a*'er each addi'ion ere observed. 9 5'a!i*a*ive C"!"r Reac*i"n)/ Mi!!"n:) Te)* In +illon1s 'es', : dros o* +illon1s reagen' as added 'o 'he dilu'ed sa%le and 'he color changes ere no'ed. 7;8 ; 5'a!i*a*ive C"!"r Reac*i"n)/ H"kin)< C"!e *e)* In 2o3ins0&ole 'es', "> dros o* 2o3ins0&ole reagen' as sloly added 'o 'he sa%les and as %i)ed ell. "> dros o* 2 "4O9  as sloly added in'o an inclined 'es' 'ube and 'he color changes ere no'ed. 7;8 = 5'a!i*a*ive C"!"r Reac*i"n)/ Saka1'c7i Te)* In 4a3aguchi 'es', !> dros o* !>? -aO2 and !> dros o* >.>"? nah'hol solu'ion ere added 'o 'he sa%les and ere %i)ed. ; %inu'es as allo''ed 'o le' i' s'and. ; dros o* "? -aO#r as added, as %i)ed and as no'ed *or changes. 7;8 > 5'a!i*a*ive C"!"r Reac*i"n)/ Ni*r"r'))i(e *e)*

In -i'rorusside 'es', >.: %( o* ;+ -aO2 as added 'o >.: %( o* sa%le. >.": %( o* "? ni'rorusside solu'ion as also added and 'he *or%a'ion o* a red solu'ion as also no'ed. 7;8 -? 5'a!i*a*ive C"!"r Reac*i"n)/ F"7!:) Te)* In 5ohl1s 'es', : dros o* ;>? -aO2 and " dros o* :? @&2 ;&OO"$b as added 'o 'he sa%les in a 'ube and as laced in a a'er ba'h. The aearance as no'ed. 7;8 -- 5'a!i*a*ive C"!"r Reac*i"n)/ Te)* ."r Ami(e) In Tes' *or A%ides, ! %( o* ">? -aO2 and !> dros o* 'he sa%le as added 'oge'her in a 'ube and as laced in a a'er ba'h. The 'es' *or evolu'ion o* gas as er*or%ed by lacing a %ois'ened red li'%us aer over 'he %ou'h o*  'he 'ube and 'he resul's ere no'ed. 7;8 -3 5'a!i*a*ive C"!"r Reac*i"n)/ %a'!0 Te)* In $auly Tes', 'he diazo reagen' as reared by %i)ing : dros o* !? sul*anilic acid i'h ; dros :? -a-O" solu'ion. : dros o* 'he sa%le and : dros o* !>? -a "&O;  'o 'he diazo reagen' and 'he aearance as no'ed. 7;8

RES#LTS AND DISC#SSION - I)"!a*i"n ". M0"1!"2in A*'er isola'ion, reddish bron i'h bee* lu%s as *or%ed. 3 Aci( H0(r"!0)i) ". In*ac* %r"*ein) A*'er hydrolysis, a clear solu'ion as *or%ed a' 'he 'o hile 'urbid a' 'he bo''o% i'h a reddish bron color. In acidic hydrolysis, 'he isola'ed %yoglobin 'he !:

7"8 &a%bell, +. ., @">!:.C">!" Biochemistry . &hinaB #roo3sC &ole, &engage (earning 7;8 &risos'o%o, &.&., Daya, +. (., e' al @">!>. Laboratory Manual in General Biochemistry . uezon &i'yB & F E $ublishing 798 essel , A. Molecules of Life h''sBCCa%i'!b.ordress.co%C'he0%olecules0o*0 li*eCabou'Ca%ino0acidsC ;C!:C">!: 7:8 -iga% A., Aryyagari #. @">>, .Lab Manual  in Biochemistry: Immunology and Biotechnology . Ta'a +cGra02ill

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