Isolation and Characterization of Proteins (Qualitative Colored Reactions and Paper Chromatography) Jenny Lynn A.

Karunungan, Daniel Adam Levy, Josell Mary Engelee im, !lphonse eandro omotan, Ma. Eloisa Mateos Group 6 2B Medical ec!nology Bioc!emistry La"oratory
#n t!e e$periment,%ualitative &olor 'eactions (as per)ormed in order to analy*e t!e c!emical goups responsi"e )or t!e color reaction o) t!e protein Gluten. Di))erent tests suc! as Biuret, +in!ydrin, ,ant!!oproteicm Millon-s, .op/ins0&ole, 1a/aguc!i, +itroprusside, 2o!l-s, est )or Amides, and 3auly yielded di))erent colored solutions as results. !e di))erent results (as due to t!e di))erence in t!e side c!ains present in Gluten. 3aper &!romatograp!y (as also conducted to "e a"le to separate and determine t!e amino acid constituents o) t!e acidic, "asic, and en*ymatic !ydrolysate o) Gluten, a protein )ound in (!eat )lour, "ased on t!e polarities o) 3roline, Alanine, &ysteine, yrosine, and Glycine. A 4.5 cm margin !ad "een dra(n across t!e longer "ottom edge o) t!e c!romatograp! paper. Eig!t e6uidistant points (ere plotted along t!e line (!ere t!e given amino acids and protein !ydrolysate samples !ad "een applied. !e L& plate (as t!en introduced in t!e solvent system consisting o) 40 Butanol,acetic acid and (ater (it! a ratio o) 78 48 5. !en, ') values )or t!e standards (ere computed and it (as determined t!at t!ere (ere 9 amino acids present in t!e acid !ydrolysate o) gluten and t!ey are 8 Alanine :') value ; <.4=>, yrosine :') value ; <.99> and an amino acid (it! a ') value o) <.79 :un/no(n since it did not matc! (it! any o) t!e used standards o) amino acids>. 2or t!e Al/aline .ydrolysate o) gluten, &ystein :') value ; <.44> and yrosine :') value ; <.97> (as identi)ied. And lastly, )or en*ymatic !ydrolysate o) gluten, yrosine :') value ; <.94> and Glycine :') value ; <.49> (as identi)ied.

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Amino Acids are molecules t!at !ave an amino group and a car"o$yl group attac!ed to its 0 car"on atom. #t-s 0car"on atom is also attac!ed to !ydrogen and a side c!ain t!at varies "et(een di))erent amino acids. !ere are 24 Amino Acids t!at are usually )ound in proteins. 3roteins, also /no(n as peptides, are organic compounds composed o) amino acids arranged in a linear c!ain. !ey are essential to li)e and participate also in cataly*ing "ioc!emical reactions, structural and mec!anical )unctions, and ot!er cell processes. 3roteins are also t!ree0 dimensional. !eir structure is comple$ t!us t!ey are de)ined in 7 terms8 primary, secondary, tertiary, and 6uaternary structure. !e primary structure de)ined t!e se6uence o) amino acids present in t!e protein, t!e secondary structure de)ines t!e arrangement in space o) t!e atoms in t!e peptide "ac/"one, t!e tertiary structure de)ines t!e 90D arrangement o) all t!e atoms in t!e protein, and t!e 6uaternary structure de)ines t!e arrangement o) su"units (it! respect to one anot!er in a protein. Gluten is a (ater insolu"le protein composite )ound in )oods processed )rom (!eat and related grain species, including "arley and rye. #t is t!e one t!at gives elasticity to doug!, !elping it rise and /eep its s!ape and o)ten gives t!e )inal product a c!e(y te$ture.?=@ Gluten is composed o) t(o maAor protein

components, glutenin and gliadin, interact in an a6ueous system to produce t!is viscoelastic property. Glutenin, t!e !ig!er molecular (eig!t protein )raction, contri"utes elasticity. Gliadin, o) lo(er molecular (eig!t, provides e$tensi"ility. #t is descri"e t!at gluten !as a polar group level o) a"out 4<B, (it! a resultant net positive c!arge (!ic! is a lo( level o) polarity o) t!e total amino acid structure compared to normal 3olar grouping levels o) 9<075B )or most )ood proteins, t!at cause t!e viscoelastic "e!avior o) !ydrated (!eat gluten t!at persists even in t!e presence o) e$cess (ater. !e result o) t!is reduced polarity is t!at e$cess (ater is repelled and t!e (!eat gluten molecules associate closely toget!er and resist dispersion. ?4<@ !is e$periment (anted to also 6ualitatively analy*e t!e amino acid components o) Gluten, and so, seeral tests (ere made to analy*e t!ese. !e tests t!at (ere involved in t!e e$periment (ere Biuret tests, +in!ydrin test, ,ant!oproteic test, Millon-s test, .op/ins0&ole test, 1a/aguc!i test, +itroprusside test, 2o!l-s test, and t!e test )or amide. 3aper &!romatograp!y (as also used to 6ualitatively separate and identi)y t!e amino acids present in t!e 9 !ydrolysates o) Gluten. 3aper &!romatograp!y is a common tec!ni6ue used to separate amino acids since it is 6uic/ and re6uires less materials. #t contains a mo"ile and a stationary p!ase. !e principle o) separation in

paper c!romatograp!y is "ased on t!eir polarity. &omponents o) t!e sample (ill separate readily according to !o( strongly t!ey adsor" on t!e stationary p!ase versus !o( readily t!ey dissolve in t!e mo"ile p!ase. #denti)ication o) t!e separated components o) un/no(n su"stance su"Aected in paper c!romatograp!y is determined "y comparing eac! ') values :relative to t!e )ront values> o) t!e separated components (it! t!e standard component o) /no(n identity. !e o"Aectives o) t!is e$periment is to primarily 6ualitatively and 6uantitatively analy*e and identi)y t!e amino acids present in t!e protein Gluten "y use o) 3aper &!romatograp!y and %ualitative colored analysis.

E)PERIME"#! !* Compounds tested (or (amples used)

!e samples used in t!is e$periment are #ntact protein Gluten and En*ymatic .ydrolysate Gluten. !e intact protein Gluten is lig!t "ro(n in color and !as a c!e(y te$ture.

'*Procedure +*Qualitative Color Reactions

en sample test tu"es o) intact protein gluten (ere prepared "y adding pinc! amount o) intact protein to 9 ml distilled (aterC and anot!er ten sample test tu"es containing en*ymatic !ydrolysate gluten (ere prepared "y adding 5 drops o) t!e !ydrolysate to 9 ml distilled (ater. Dne out o) ten sample test tu"es o) intact protein and !ydrolysate are su"Aected to di))erent test8 'iuret #est, 2< drops o) 2.5 M +aD. (ere added to eac! sample test tu"es o) intact protein and en*ymatic !ydrolysate and t!en 209 drops o) <.4 M &u1D7 solution (ere added. Bot! test tu"es (ere s!a/en and t!e color (as noted. "inhydrin #est, 604< drops o) <.4B nin!ydrin solution (ere placed to eac! sample test tu"es o) intact protein and en*ymatic !ydrolysate. 1ample test tu"es (ere t!en !eated in a "oiling (ater "at! and appearance o) "lue violet coloration (as to "e noted. )anthoproteic #est, #n eac! test tu"e o) intact protein and en*ymatic !ydrolysate, 4< drops o) conc. .+D9 (ere slo(ly added, t!en mi$ed (ell and t!e color (as noted. Anot!er 4< drops o) conc. +aD. (ere added slo(ly and t!e color (as noted also. Million-s #est, 2ive drops o) Million-s reagent (ere added to t!e diluted samples o) intact protein and en*ymatic !ydrolysate and t!e color (as noted. .op/ins0Cole #est, #n eac! diluted samples, 2< drops o) .op/ins0&ole reagent (ere added and (ere mi$ed (ell. !en eac! diluted samples are inclined and slo(ly added along t!e side (it!

2< drops conc. .21D7. Do not s!a/e t!e mi$ture and note t!e color at t!e inter)ace. (a/aguchi #est, en drops o) 4<B +aD. and 4< drops o) <.<2B nap!t!ol solution (ere added to eac! diluted samples o) intact protein and en*ymatic !ydrolysate, mi$ed (ell and let stand )or 9 minutes. !en 9 drops o) 2B +aDBr (ere added and t!e color produced (as noted. "itroprusside #est, #n eac! diluted samples, <.5 ml o) 9 M +aD. and <.25 ml 2B nitroprusside solution (ere added t!en )ormation o) red solution (as noted. 1ohl-s #est, 5 drops o) 9<B +aD. and 2 drops o) 5B :&.9&DD>23" (ere added to one o) eac! diluted samples o) intact protein and en*ymatic !ydrolysate t!en placed in a "oiling (ater "at! and noted t!e appearance o) dar/:"lac/ or "ro(n> sediment. #est for !mides, 4 ml o) 2<B +aD. (as added to eac! diluted samples o) intact protein and en*ymatic !ydrolysate, t!en (as placed in a "oiling (ater "at! and test )or evolution o) gas (!ile !eated (as noted "y placing a moistened litmus paper. !e color o) t!e litmus paper (as noted. Pauly #est, !e diluted samples o) intact protein and en*ymatic !ydrolysate and 9 drops o) 4< B +a2&D9 (ere added to t!e dia*o reagent :contains 905 drops o) 4B sul)anilic acid and 9 drops o) 5B +a+D solution>. !e appearance o) red coloration (as noted.

2*Paper Chromatography (et0up

Dn a paper c!romatogram, an origin (as dra(n (it! a pencil line 4.50cm )rom t!e "ottom o) t!e longer edge o) t!e c!romatogram. 49 e6uidistant points (ere mar/ed on t!e line )or spotting o) t!e amino acid standards and t!ree !ydrolysate samples. !e standards (ere applied )ive times,and t!e samples ten times using capillary tu"es. !e sample (as allo(ed to dry "et(een applications. !e c!romatogram (as placed inside t!e 4<<< mL "ea/er (!ic! (as used as a c!am"er. !e level o) t!e solvent (as "elo( t!e origin. !e "ea/er (as covered (it! a 3etri dis!. A)ter appro$imately 9< minutes t!e c!romatogram (as removed and t!e solvent )ront (as mar/ed (it! a pencil. !e c!romatogram (as air dried, and t!en 4B nin!ydrin reagent (as dropped on t!e (!ole sur)ace o) t!e c!romatogram. !e c!romatogram (as once again dried, revealing t!e amino acid spots. !e densest areas o) t!e spots (ere t!en encircled, and t!e ') values (ere t!en computed.

RE(& #( !"% %I(C&((I$"

Q&! I#!#I3E C$ $R RE!C#I$"( !e e$periment analy*ed t!e c!emical groups responsi"e )or color reactions and (it! t!e

principle o) eac! tests, 6ualitative analysis (as done. 1. 'iuret #est !e Biuret test detects peptide "onds 0 and turns violet :positive result> in t!e presence o) proteins. !e Biuret reagent is made o) sodium !ydro$ide and copper sul)ate. Gluten rendered a positive result ?6@. 2. "inhydrin #est Amines, or alp!a0amino acids react (it! nin!ydrin to yield a colored result. Gluten, li/e alp!a0amino acids reacted to t!e test and yielded a lig!t violet coloration 0 suggesting a positive result ?9@. 4. )anthoproteic #est A $ant!oproteic reaction is t!e reaction o) tyrosine0containing proteins (it! nitric acid 0 turning t!e product yello(. !e $ant!oproteic test is carried out "y adding concentrated nitric acid to t!e su"strate "eing tested. #) proteins are present containing amino acids (it! aromatic rings 0 t!e mi$ture turns yello( :li/e in t!e case o) Gluten> ?7@. 5. Millon6s #est !is test detects t!e presence o) solu"le proteins. !e color produced is given "y derivatives o) "en*ene in (!ic! a !ydrogen !as "een replaced "y a !ydro$yl group. !e reaction serves as a test )or t!e presence o) tyrosine, trypt!opane, and p!enylalanine. A positive result (ould yield a reddis!0"ro(n color ?5@. 7* .op/ins0Cole #est !is test determines t!e presence o) t!e amino acid tryptop!an 0 (!ic! creates a violet ring (!ere t(o layers meet ?E@. 8* (a/aguchi #est !is test is used :in t!is case> to detect arginine, and reacts (it! t!e guanidine group o) t!e protein to give a red color. Gluten tested negative )or arginine ?4, 2@. 9* "itroprusside !is test is speci)ic )or cysteine, t!e only amino acid containing a sul)!ydryl group :01.>. !e gluten tested negative )or cysteine ?2@. :* 1ohl6s #est !is test determines i) t!e protein !as sul)ur containing amino acids, li/e cysteine, cystine, and met!ionine. A positive result (ould "ring a"out a red decolori*ation. ;* #est for !mides !e c!anging o) t!e litmus paperFs color )rom red to "lue indicates a presence o) a "asic componentG"asic amino acid in gluten. +<* Pauly6s #est !e imida*ole group reacts (it! dia*oti*ed aulp!anilic acid to )orm !ig!ly colored

a*ocompounds. #n al/aline medium t!is is red in color ?H@. &olor 'eaction Biuret +in!ydrin ,ant!oproteic Millon-s .op/ins0&ole #ntact Gluten Lig!t violet coloration :I> &olorless :0> Jello( to orange coloration :I> K!ite or slig!tly tur"id solution &olorless :0> En*ymatic .ydrolysate &olorless :0> &olorless :0> &olorless :0> &olorless :0> &olorless or no layer )ormed :0> &olorless :0> Jello( 1olution :0> &olorless :0> &olorless :0>

1a/aguc!i +itroprusside 2o!l-s est Amide )or

&loudy solution Jello( solution :0> Bro(n0"lac/ sediment :I> Lig!t pin/ coloration,negative in "lue ltmus paper :I> Dar/ yello( or orange coloration :0>

3auly

Jello( solution :0>

#a=le +* %ualitative &olor 'eaction 'esults P!PER C.R$M!#$>R!P.? 3aper &!romatograp!y is t!e tec!ni6ue used to 6ualitatively analy*e t!e acidic, "asic and en*ymatic !ydrolysates o) Gluten, a protein )ound in (!eat )lour. !is tec!ni6ue is )acilitated "y t!e principle o) polarity to(ard t!e t(o p!ases, t!e stationary and t!e mo"ile p!ase. Amino Acid 1tandard 3roline &ysteine yrosine ') Lalues o) t!e 1pots
1tandard .I .ydrolysa te D.0 .ydrolysa te En*ymatic .ydrolysa te

<.2E <.4< <.94

0 0 <.99

0 <.44 <.97

0 0 <.94

Glycine Alanine

<.49 <.2<

0 <.4=

0 0

<.49 0

#a=le 2* 3aper &!romatograp!y 'esults !e e$periment utili*ed a 42$45 c!romatograp! paper plotted (it! 5 di))erent amino acid standards :Alanine, Glycine, &ysteine, 3roline and, yrosine> and 9 !ydrolysates o) Gluten :acid, "asic and, en*ymatic> along t!e line o) its origin located 4.5 cm a"ove its "aseline. !e c!romatograp! paper (as placed inside a pre0e6uili"rated c!am"er consisting o) 40 Butanol,acetic acid and (ater (it! a ratio o) 78 48 5. A !ig!er ') value (ould mean t!at t!e su"stance "eing identi)ied is less polar or non0 polar (!ile a lo(er ') value (ould mean ot!er(ise. Msing t!e ta"le a"ove, it (as made clear t!at &ysteine and Glycine !ad a lesser a))inity to t!e mo"ile p!ase since it !as a lesser ') value and t!us proving t!at it is a polar unc!arged amino acid. Dn t!e ot!er !and, Alanine and 3roline !ad a !ig! ') value, it !ad greater a))inity to t!e mo"ile p!ase and t!us proving t!at it is a non0polar !ydrop!o"ic amino acid. As stated earlier, t!e solvent )ront (as composed o) 9 types o) molecules and all o) t!em !ad t!eir o(n purpose. Kater served as t!e stationary p!aseC 40Butanol, a non0polar molecule carried t!e non0polar amino acids up to t!e mo"ile p!ase andC Acetic acid, a polar molecule t!at carried t!e polar amino acids up to t!e mo"ile p!ase. 1ince t!ere (as a 784 ratio o) 40Butanol and Acetic acid it could "e said t!at non0polar amino acids are )avored t!an t!e polar ones t!us, non0polar amino acids li/e Alanine and 3roline !ad a !ig!er ') value t!an t!e polar amino acids &ysteine and Glycine. !ese interpretations (ere made possi"le "ecause o) 4B +in!ydrin reagent. 1ince t!e 2< standard amino acids (ere all colorless, t!e c!romatograp! paper (as sprayed evenly (it! +in!ydrin reagent to ma/e t!e spots appear "lue, yello( or purple. Lastly, "y comparing t!e ') values o) t!e standards (it! t!e ') values present in t!e !ydrolysates, it (as determined t!at Alanine, yrosine and an un/no(n amino acid (it! a ') value o) <.79 (as present in t!e acidic !ydrolysate o) gluten. 2or t!e "asic !ydrolysate, &ysteine and yrosine (as present (!ile )or t!e en*ymatic !ydrolysate, yrosine and Glycine (as present.

1igure +* 3aper &!romatogram 'esult RE1ERE"CE(

?4@ Ale$ander, D. Sakaguchi Test Amino Acid Proteins. !ttp8GG(((.re)erence.comGmoti)GscienceGsa/aguc !i0test0amino0acid0proteins 4G42G47 ?2@ Boga*ici Mniversity. Experiment 2 Qualitative Analysis of Amino Acids and Proteins. !ttp8GG(((.c!em."oun.edu.trG(e"pagesGcourses G&!em745G&!emB2<745B2<E$periment B2<2.pd) 4G42G47 ?9@ .unt, #. inhydrin Test. !ttp8GG(((.c!em.ucalgary.caGcoursesG954G&arey 5t!G&!2HGc!2H0909.!tml 4G42G47 ?7@ Merriam Ke"ster Dictionary. !anthoproteic "eaction. !ttp8GG(((.merriam0 (e"ster.comGdictionaryG$ant!oproteic B2<reaction 4G42G47 ?5@ +orris, J. Experimental #rganic $hemistry Proteins. !ttp8GG(((."oo/s0a"out0 cali)ornia.comG3agesGE$perimentalNDrganicN&!e mistryGE$NDrganicN&!emN&!apN26.!tml 4G42G47 ?6@ D!io Mniversity Department o) Biological 1ciences. %iuret Test. !ttp8GG(((."iosci.o!iou.eduGintro"iosla"GBios4H< G4H<N2G"iuret.!tm 4G42G47 ?H@ 1airam, +. $olor "eactions of Proteins. !ttp8GG6ui*let.comGEE<4H2=Gcolor0reactions0o)0 proteins0)las!0cardsG 4G42G47 ?E@ :Aut!or Mn/no(n> Protein&s Test. !ttp8GG)ulltimes.(ordpress.comGproteinG 4G42G47 ?=@Ki/ipedia.'luten. !ttp8GGen.(i/ipedia.orgG(i/iGGluten 42G9<G2<49 ?4<@ E$cerpt )rom K!eat Gluten8 A atural Protein for the (uture - Today "y t!e #nternational K!eat Gluten Association !ttp8GG(((.mp"io.comGdetailedNin)o.p!pO )amilyN/ey;<24<4E45Pcountry;46E 42G2=G2<49