ISOLATION AND CHARACTERIZATION OF PROTEINS (ACID HYDROLYSIS OF MYOGLOBIN)

Group # 7, 2BMT, Mary Grace Saba, *Miguel M. Sabillena, Regine San Jose, David Santos, Sebastian irli!"nne

ABSTRACT
Myoglobin is a vital protein #or o$ygen transport in vertebrates. %t is present in large concentration in &uscle and is responsible #or t'e red color o# t'e organ. Myoglobin is e$tracted and 'ydroly(ed by acid and sub)ected to di##erent *ualitative color tests. +aper c'ro&atograp'y 'ad been done to analy(e t'e di##erent a&ino acid co&ponents o# t'e protein.

INTRODUCTION
+roteins can be considered as poly&ers o# a&ino acids. "&ino acids are lin,ed by covalent peptide bond into linear c'ain, -'ic' is called peptide or polypeptide c'ain. T'e properties co&&on to all a&ino acids are due to t'e relative special arrange&ents o# t'e carbo$yl and a&ino groups. T'e p'ysical and c'e&ical properties uni*ue to eac' a&ino acid are t'e result o# t'e structure and c'e&ical properties o# t'e R group. "&ino acids 'ave a great variety o# c'e&ical reactive groups providing a -ide range o# reactivity proteins. T'e reactions #or individual side!c'ain radicals and .!a&ino and .!carbo$yl groups are speci#ic bot' #or #ree a&ino acids and proteins, -'ereas t'e reaction #or peptide group is c'aracteristic o# t'e protein and peptides. Speci#ic reactions are used #or t'e purpose o# identi#ying a&ino acids and proteins in biological &edia, #or *ualitative and *uantitative analysis. Biuret reaction -'ic' accounts #or deter&ining peptide bonds, /in'ydrin reaction is typical #or .!a&ino acids. 0ant'roproteic test detect side!c'ains o# aro&atic a&ino acids and Millon1s and 2op,ins! 3ole tests are #or t'e deter&ination o# tyrosine and tryptop'an residues, respectively. 4o'l1s test is used to ,no- i# sul#ur!containing a&ino acids are present and sul#osalicylic acid #or t'e i&ida(ole ring o# 'istidine residues. T'e 'ydrolysis o# a&ides under acidic condition is basically t'e sa&e as protein 'ydrolysis, because peptide bond lin, is t'e sa&e as an a&ide lin,. %# you scale t'is up to a polypeptide c'ain, eac' o# t'e peptide lin,s -ill be bro,en in e$actly t'e sa&e -ay. T'at &eans it -ill end up in a &i$ture o# t'e a&ino acids t'at constitute t'e protein. +rotein 'ydrolysates can be tested *ualitatively by di##erent reagents and as -ell as paper c'ro&atograp'y. +artition paper c'ro&atograp'y is -idely e&ployed #or t'e deter&ination and separation o# a&ino acids. T'e solvent &igrates along a strip o# paper and carries a&ino acid dissolved in it.

EXPERIMENTAL Acid Hydrolysis of Intact Protein

Myoglobin e$tracted #ro& ground bee# -as 'ydroly(ed by acid a#ter it -as tested -it' di##erent *ualitative color tests. T'e 'ydrolysis process undergone by adding 5 &6 o# 7 / sul#uric acid to 8.5 gra&s o# intact protein and t'en autoclaved #or 5 'ours 9:5 psi;. Ten &6 o# distilled -ater -as added to t'e autoclaved sa&ple and t'en trans#erred to a 258 &6 bea,er. "#ter t'e sa&ple -as trans#erred, 5&6 o# 7/ bariu& 'ydro$ide -as added and t'en &i$ed. T'e sa&ple -as neutrali(ed by adding 7/ bariu& 'ydro$ide or 7/ sul#uric acid. %t -as t'en tested -it' red and blue lit&us paper to c'ec, i# it -as already neutral. T'e sa&ple -as #iltered and t'e #iltrate -as collected #or c'aracteri(ation test and paper c'ro&atograp'y. Qualitative Color Reactions T'e &yoglobin 'ydrolysate -as tested -it' di##erent c'aracteri(ation reagents na&ely< Biuret, /in'ydrin, 0ant'roproteic, Millon, 2op,ins!3ole, Sa,aguc'i, 4o'l, and Dia(o tests.

=ig't test tubes -ere prepared #or eac' o# t'e test reaction. =ac' test tube consisted o# : &6 o# distilled -ater added to 8.5 &6 o# 'ydroly(ed sa&ples. %n Biuret test :8 drops o# t'e Biuret reagent -as added to one test tube, s'a,en and t'e observed #or color c'anges. 4or /in'ydrin test, >!:8 drops o# 8.: ? /in'ydrin solution -as added to t'e dilute sa&ple and 'eated in a -ater bat'. T'e appearance o# a blue!violet coloration -as ta,en note o#. %n 0ant'roproteic test, concentrated nitric acid and concentrated sodiu& 'ydro$ide :8 drops eac' -ere added slo-ly and t'en &i$ed. 3olor c'anges a#ter eac' addition -ere observed. 4ive drops o# Millon1s reagent -as added to t'e diluted sa&ple -it' t'e color ta,en note o#. 4or 2op,ins!3ole test, 28 drops o# 2op,ins!3ole reagent -as slo-ly added to t'e sa&ple and &i$ed -ell. Sul#uric acid about 28 drops -as slo-ly added along side t'e inclined test tube. T'e color o# t'e inter#ace -as ta,en note o#. "#ter t'e addition o# :8 drops o# Sa,aguc'i reagent and &i$ed, it -as let stand #or @ &inutes and @ drops o# 2? 'ypobro&ite -as added. " red color indicated t'e presence o# arginine in t'e protein. T'e addition o# 28 drops o# >M sodiu& 'ydro$ide and a #e- crystals o# lead acetate to t'e diluted sa&ples provided dar, bro-n sedi&ent during boiling o# t'e sa&ple provided t'e positive result in 4o'l1s reaction. 6astly, a preparation o# @!5 drops o# :? sul#osalicylic acid -it' @ drops o# 5? sodiu& nitrite -as added to 5 drops o# t'e diluted sa&ple and 5 drops o# :8? sodiu& carbonate solution. " red coloration -as observed. Separation and Identification of Amino Acid by Paper Chromatography " :.5 c& &argin #ro& one edge o# t'e paper -as &easure and dra-n lig'tly -it' a pencil on t'e prepared :2 $ 28 c& A'at&an #ilter paper # :. Ten e*uidistant points -ere labeled on t'e line #or application o# t'e sa&ples. T'e sa&ples -ere air!dried bet-een applications by a capillary tube. T'e paper -as t'en rolled into a cylinder -it'out overlapping and t'en stapled. %t -as t'en placed on t'e pre!e*uilibrated c'a&ber and t'en covered #or t'e solvent to ascend. A'en t'e solvent reac'ed at least 2 c& #ro& t'e ot'er end, t'e paper -as re&oved and t'e solvent #ont -as i&&ediately &ar,ed. T'e paper -as air!dried and sprayed lig'tly -it' nin'ydrin reagent. T'en it -as oven!dried and observed #or t'e a&ino acids t'at appeared as blue, purple and yello- spots on t'e paper.

T'e spot -as encircled and co&puted #or t'e R# values.

RESULTS AND DISCUSSIONS

Qualitative Color Test for Hydrolyzed yoglobin

%n Table : t'e c'aracteri(ation o# t'e 'ydroly(ed protein, data results o# intact protein o# t'e sa&e tests -ere co&pared -it' t'e #or&er to s'o- t'e di##erence o# t'e reactions in c'aracteri(ation. Table 1. Results of Qualitative Color Reaction of Intact and Hydrolyzed Proteins Color Reac !o" I" ac H#$rol#%e$ Pro e!" Pro e!" B!&re Blue 3olorless N!"'#$r!" 6ig't bro-n B3olorless Xa" 'ro(ro e!c Blue!green B3olorless M!llo")* 4les' B3olorless Ho(+!"*,Cole 6ig't bro-n B3olorless Sa+a-&c'! Red 4les' Fo'l)* Blac, Bro-n D!a%o reac !o" Red B3olorless BFalse results due to some technical errors in the experiment. Cnly t'e 4o'l1s and Sa,aguc'i test provided a positive result #or t'e color c'aracteri(ation o# t'e 'ydroly(ed &yoglobin indicating t'e presence o# arginine and sul#ur containing a&ino acid 9cysteine and &et'ionine;. 3o&pared -it' results o# t'e c'aracteri(ation test o# t'e intact &yoglobin, only t'e /in'ydrin test provided a negative result indicating t'e absence o# .!a&ino acid in t'e intact protein. Paper Chromatography Analysis of yoglobin

3'aracteri(ation o# &yoglobin using paper c'ro&atograp'y yielded di##erent and inco'erent results co&pared -it' t'e colored c'aracteri(ation o# &yoglobin 'ydrolysate. T'e R# values o# standards and t'e 'ydrolysate listed in Table 2 provided t'e additional protein result bet-een t'e t-o *ualitative tests.

Table . Rf values of the amino acid standards and hydrolyzed myo!lobin. R/ 0al&e* o/ 'e S(o * A.!"o Ac!$ Ac!$ S a"$ar$* S a"$ar$* H#$rol#*a e Tr#( o('a" 8.>@ 8.>@ Ar-!"!"e 8.5: 8.D7 Prol!"e 8.7@ C#* e!"e 8.@> Ser!"e 8.>@ 8.>@ A*(ar !c Ac!$ 8.@D T#ro*!"e 8.72 H!* !$!"e 8.>8 8.>@ Gl#c!"e 8.5E 8.>@ Ala"!"e 8.>7 Tryptop'an, arginine, serine, 'istidine, and glycine are separated and identi#ied co&ponents o# &yoglobin. T'e presence o# arginine in paper c'ro&atograp'y supported t'e results o# t'e color c'aracteri(ation tests o# t'e 'ydroly(ed protein t'at is c'aracteri(ed by t'e Sa,aguc'i test. T'e presence o# tryptop'an, serine, 'istidine and glycine -ere strengt'ened by t'e positive results o# t'e intact &yoglobin -it' t'e di##erent *ualitative colored test. Test discussion and comparison T'e sub)ection o# &yoglobin in acid 'ydrolysis -'ic' is co&posed o# e$tre&e p2 and te&perature -'ic' are co&&on denaturing agent o# protein destroyed t'e peptide bonds o# t'e protein leaving t'e .!a&ino acids susceptible and #ree #or reactions. %t is t'e pri&ary reason -'y t'e Biuret reaction -as negative #or t'e 'ydroly(ed protein because Biuret reagent deter&ines t'e peptide bond and *uantity o# a protein. /in'ydrin reaction is negative #or t'e intact protein -'ile positive #or t'e 'ydroly(ed &yoglobin due to t'e presence o# t'e .!a&ino acid in t'e latter. Based #ro& resources, all o# t'e 28 essential a&ino acids are co&ponents o# &yoglobin e$cept #or gluta&ine and asparagine -'ic' are not standards o# t'e paper c'ro&atograp'y test. T'e errors o# t'e e$peri&ent could be t'e cause o# tec'nical and personal errors o# t'e e$peri&enter. 0ant'roproteic reaction is speci#ic #or cyclic a&ino acids suc' as p'enylalanine, tyrosine, tryptop'an and 'istidine. "ro&atic a&ino acids react -it' nitric acid, yielding a yellonitroco&pound, -'ic' c'anges color to orange in al,aline &ediu& o-ing to t'e #or&ation o# salt positive #or t'e basic 'ydrolysis.

Since 'istidine and tryptop'an are aro&atic a&ino acids, 0ant'roproteic reaction yielded a positive result #or t'e intact protein but not #or t'e 'ydrolysate caused probably by t'e errors or loss o# protein during 'ydroly(ation process. Millon1s reagent is used #or t'e deter&ination o# tyrosine content in proteins. %t is co&posed o# salts o# &ercury dissolved in nitric acid. Tyrosine 9bot' #ree and constitutive o# proteins; reacts -it' reagent and produces a purple!red salt o# &ercury.

Millon1s test is positive #or t'e intact protein but not in t'e 'ydroly(ed &yoglobin 9also negative result in paper c'ro&atograp'y; caused probably by t'e sa&e reason as in 0ant'roproteic reaction. Dia(o reaction is typical #or proteins containing cyclic a&ino acids suc' as tyrosine and 'istidine. T'e a&ino acids produce a red colored dia(o dye in reaction -it' t'e dia(o reagent and t'e intensity o# t'e color depends on t'e a&ount o# cyclic a&ino acid present.

%n t'e intact &yoglobin, t'e dia(o reaction provided a positive result -'ile negative #or t'e 'ydroly(ed &yoglobin caused probable by t'e sa&e errors and reason o# t'e &illon1s and $ant'ropeoteic reaction. Sa,aguc'i reaction is typical #or arginine only. "rginine reacts -it' .!nap'tol and produces a red colored derivative. T'e presence o# arginine is positive #or bot' t'e intact and 'ydroly(ed &yoglobin and supported by t'e presence o# arginine in t'e paper c'ro&atograp'y #or t'e 'ydroly(ed protein. 4o'l1s reaction is used #or deter&ination o# S! containing a&ino acids. 2eating o# &yoglobin solution in an al,aline &ediu& leads to t'e #or&ation o# sodiu& sul#ide i# t'e protein contained sul#ur a&ino acids suc' as cysteine, cystine, or &et'ionine. %n #urt'er reaction o# sodiu& sul#ide -it' lead acetate a dar, bro-n colored precipitate is #or&ed.

Rf " #distance traveled by a substance$ % #distance traveled by the solvent$

Basically, co&ponents in t'e paper t'at 'ave t'e sa&e R# values are t'e sa&e a&ino acids. Tryptop'an, arginine, serine, 'istidine, and glycine standards 'ad t'e sa&e R# value -it' t'e separated acid 'ydrolysate o# &yoglobin indicating t'eir presence in &yoglobin. "s co&pared -it' -'at t'e actual co&ponents o# &yoglobin, all o# t'e standards are present in t'e protein and t'e negative results o# t'e ot'er a&ino acid co&ponents could be t'e cause o# t'e tec'nical aspects as -it' t'e e$peri&enter errors.

REFERENCES
3a&pbell, MaryG 4arell S'a-n. 92887;. !iochemistry 9>t' ed.;. 3anada< Broo,sH3role. T'e Bioc'e&istry Depart&ent 92887;. "aboratory anual in #eneral !iochemistry$ Manila< Iniversity o# Santo To&as.

4o'l1s test is bot' positive #or intact and 'ydroly(ed &yoglobin indicating t'e presence o# cysteine in t'e protein alt'oug' t'e result o# paper c'ro&atograp'y yielded a negative result #or t'e 'ydroly(ed &yoglobin. 3'ro&atograp'y is a tec'ni*ue t'at separates &i$tures into t'eir individual co&ponents #or e$a&ple< i# -e put blac, -as'able in, onto a tissue, t'e in, -ill spread out-ards #ro& t'e place -'ere -e blotted it 'o-ever, t'e various co&ponents o# t'e in, canFt all &ove at t'e sa&e speed as it spreads out ! so t'e co&ponents -ill visibly separate. T'e principle involved in t'e separation o# t'e a&ino acid co&ponents o# t'e paper is polarity. T'e solvent syste& used 9&obile p'ase; et'anol, -ater, and a&&onia 978<:8<:8; is non!polar by nature and t'e &ore polar t'e side group, t'e #art'er it travelled as -it' proline, tryptop'an, and tyrosine. T'e &ore polar a&ino acids aspartic acid and cysteine 'ave t'e least distance travelled seen in t'e R# values o# t'e a&ino acid standard. T'e t'ing t'at is &easured in c'ro&atograp'y is t'e di##erence bet-een 'o- #ar a substance 9#ro& t'e &i$ture; travels co&pared to 'o- #ar t'e solvent travels.

Retrieved #ro& t'e Aorld Aide Aeb< 3lar,, J. 9288D;. T'e 'ydrolysis o# protein. Retrieved January :8, 288E #ro& 'ttp<HH---.c'e&guide.co.u,Horganicpro psHa&inoacidsHprotein'ydrolysis.'t&l.

%vanovienJ, 6., Mor,Kniene, R., BanienJ, R., %vanovas, 6. L BorutaitJ, . Retrieved January :8, 288E #ro& 'ttp<HH---.,&u.ltHnscHbioc'e&i)aH6abor atoryM&anualM+"RT?28%.pd#