Biology 2542 Lab TOPICS TO BE COVERED:

LAB #11

(11th/19 Lab Sessions for Winter Quarter, 2008)

Intro u!tion" W#y en$y%es are nee e to igest your foo & 'es!ri(tion of t#e !#e%i!al !o%(osition of !arbo#y rates, li(i s, an (roteins )erfor% !#e%i!al tests *naly$e results of !#e%i!al tests DESIRED OUTCOMES: After complet !" the #ct $ t e% &e%cr 'e& for th % l#' %e%% o!( %t)&e!t% %ho)l&: *U!&er%t#!& the '#% c chem c#l pr !c ple% of !)tr e!t m#cromolec)le c#t#'ol %m *Be #'le to &e%cr 'e +h, e!-,me% #re )%e& to & "e%t the foo& +e e#t *Be #'le to %)mm#r -e chem c#l compo% t o! of #!& & "e%t o! of prote !% c#r'oh,&r#te%( #!& f#t% *Be #'le to &e%cr 'e e#ch chem c#l te%t performe& o! the %)'%tr#te% #!& #!#l,-e the re%)lt% of e#ch e.per me!t MATERIALS /EEDED: Water Bat# (#eate to +,-.) /it# t#er%o%eter 0est tube ra!1s at ea!# Lab Ben!# Wa2 3ar1ing (en!ils Bunsen burners 4ing stan s 0est 0ubes Safety glasses (or one5s o/n glasses) 6our 250 %l bea1ers an four 50 %l bea1ers 0e%(late sets of 2 %l, + %l, 5 %l, an 70 %l test tube /ater 8blan1s9 *sbestos (a s :ot;#an gri((ers 0est;tube #ol ers S(e!ifi! ite%s for 0ests an )ro(erties of .arbo#y rates" 5< fru!tose solution 5< glu!ose solution 70< glu!ose solution 0#in star!# (aste Bene i!t5s solution Barfoe 5s reagent 7 3 :.l 7 3 =a>: S(e!ifi! ite%s for 0ests an )ro(erties of Li(i s" 5< )an!reatin solution Blue lit%us solution S/eet !rea% (fres#) 5< =a:.>+ S(e!ifi! ite%s for 0ests an )ro(erties of )roteins" @n ilute egg /#ite 7< albu%in solution (egg /#ite) 7< .uS>4 70< =a>: 0A7< al!o#ol solution of gly!ine (or alanine) Buffer solution ((:8) 5< )an!reatin solution Woo en stirring sti!1s 5< su!rose solution Seli/anoff5s reagent Lugol5s io ine (I?I)

Act $ t, #1: Intro u!tion" W#y are en$y%es nee e to igest your foo &
Before nutrients !an be !onBerte to usable energy by !ells, t#e large organi! macromolecules in foo %ust be catabolized into monomers, t#e buil ing blo!1s of %a!ro%ole!ulesA 0#ese !#e%i!al rea!tions are !on; trolle by enzymes produced by the digestive system. Enzymes are (rotein (organi!) catalysts t#at lo/er acti; vation energy, t#e energy reCuire for a !#e%i!al rea!tionA Wit#out en$y%es, t#e bo y /oul #aBe to #eat u( to angerous te%(eratures to (roBi e t#e a!tiBation energy ne!essary to e!o%(ose ingeste foo A Dn$y%es #aBe

Biology 257 LAB #11 !ontinue

)age 0/o

a narro/ range of (#ysi!al !on itions in /#i!# t#ey o(erate at %a2i%u% effi!ien!yA Temperature an pH are t/o i%(ortant fa!tors of en$y%ati! rea!tionsA 6or e2a%(le, en$y%es inBolBe in (rotein igestion reCuire Cuite a ifferent (: (aroun 2A2 2A+) t#an /#at is nee e for !arbo#y rate igestion (aroun 8)A 0#e en$y%e (e(sin is %ost a!tiBe in t#e a!i i! !on itions of t#e sto%a!#A 0#e ne2t (rotein; igesting en$y%e in t#e seCuen!e, try(sin, reCuires an al1aline enBiron%entA *n en$y%ati! rea!tion inBolBes reactants, !alle t#e substrate, an results in one (or %ore) pro&)ct(s)A 0#e en$y%e itself #as an active site /#ere t#e substrate bin sA >nly a substrate t#at is !o%(atible /it# an en; $y%e5s a!tiBe site is %etaboli$e , an t#e en$y%e is sai to #aBe specificity for !o%(atible substratesA @(on .o%(letion of t#e !#e%i!al rea!tion, t#e (ro u!t is release , an t#e en$y%e, unaltere in t#e rea!tion, !an bin to anot#er substrate %ole!ule an re(eat t#e rea!tion %any ti%es in su!!essionA Da!# en$y%e #as a narro/ range of (#ysi!al !on itions in /#i!# it o(erates at %a2i%u% effi!ien!yA 0e%(erature an (: are t/o i%(ortant fa!tors in en$y%ati! rea!tionsA * goo e2a%(le of en$y%e !on itions are t#ose reCuire in t#e (rotein igestion e2(eri%ent in t#is e2er!iseA 0#e sto%a!# (ro u!es an ina!tiBe en; $y%e, a proenzyme, !alle (epsinogenA W#en (e(sinogen is %i2e /it# sto%a!# a!i , t#e (roen$y%e is !on; Berte into its a!tiBe (rotein; igesting en$y%e for%, pepsin, /#i!# begins to !ataboli$e large (rotein %ole!ules in t#e foo A W#en %aterials fro% t#e sto%a!# %oBe into t#e s%all intestine, t#e (an!reas releases so iu% bi!arbonate /#i!# buffers t#e sto%a!# a!i an raises t#e a%bient (: to , 8A Be!ause (e(sin is a!tiBe only at an a!i i! (:, it be!o%es less a!tiBe in t#e s%all intestine /#ile ot#er en$y%es ta1e t#eir res(e!tiBe turns %eta; boli$ing t#e substrateA Li1e all (rotein %ole!ules, an en$y%e5s fun!tion is relate to its stru!tural s#a(e, %u!# as t#e s#a(e of a 1ey eter%ines /#i!# lo!1 it fitsA So%e en$y%e e2(eri%ents in t#is e2er!ise /ill be in!ubate in a /ar%;/ater bat# set at bo y te%(erature, +,-.A 0oo #ig# a te%(erature enatures an en$y%e, !ausing a !#ange in s#a(e, an estroys t#e en$y%eA Eour igestiBe syste% !ataboli$es t#e foo you ingest /it# a !o%(le2 seCuen!e of en$y%esA If an en$y%e is absent or se!rete in an insuffi!ient Cuantity, t#e substrate !annot be igeste A So%e in iBi uals, for e2a%(le, o not (ro u!e t#e en$y%e la!tase /#i!# igests la!tose, !o%%only !alle 8%il1 sugar9A If a la!tose; intolerant in iBi ual !onsu%es airy (ro u!ts, /#i!# are #ig# in la!tose, t#e sugar re%ains in t#e istestiBe tra!t an is only Bery slo/ly igeste by ba!teriaA 0#is results in gas, intestinal !ra%(s, an iarr#eaA Dn$y%es as a grou( are inBolBe in bot# !ataboli! ( e!o%(osition) an anaboli! (synt#esis) rea!tionsA * s(e!ifi! en$y%e, #o/eBer, fun!tions in only one ty(e of rea!tionA Dn$y%es of t#e igestiBe syste% generally !ause !ataboli! rea!tions to %etaboli$e your foo into s%aller %ole!ules /#i!# !an !ross !ell %e%branes an su((ly ra/ %aterials for !ellular res(irationA 'igestion in!lu es bot# mechanical digestion, %e!#ani!ally brea1ing foo into s%aller (ie!es, an chem.ical digestion, t#e en$y%ati! brea1 o/n of large %ole!ules into s%aller ones t#at !an be absorbe into t#e bloo strea%A Mechanical digestion begins in t#e %out# an !ontinues in t#e sto%a!# an s%all intestineA .#e/ ing, or mastication, brea1s o/n large (ie!es of foo into s%aller ones t#at are s/allo/e A In t#e sto%a!#, (eri; stalti! /aBes %e!#ani!ally !#urn an %i2 t#e foo /it# gastri! Fui!es to en#an!e igestionA .#urning !ontinues in t#e s%all intestine by s%oot# %us!le !ontra!tions !alle segmentations, or ba!1 an fort# %oBe%ents of (or; tions of t#e s%all intestine, t#at %i2 t#e !#y%e /it# igestiBe en$y%es (rior to (eristalti! !ontra!tions %oBing t#e !ontents alongA Mechanical digestion en#an!es t#e s(ee of !#e%i!al igestion by !reating a greater surfa!e area for igestiBe en$y%es to o t#eir /or1A Chemical digestion inBolBes igestiBe en$y%es brea1ing t#e bon s of t#e foo %a!ro%ole!ulesA 0#e foo /e eat !ontains %a!ro%ole!ules of !arbo#y rates, li(i s, an (roteinsA (It !ontains t#e nu!lei! a!i s 4=* an '=*, too, #o/eBer, /e on5t usually t#in1 of t#ese as 8nutrient9 %ole!ulesG) Be!ause t#ese %ole!ules are too large to be absorbe as is by t#e gastrointestinal tra!t, en$y%es se!rete by Barious (arts of t#e igestiBe sys;te% catabolize (brea1 o/n) large %ole!ules into s%all ones t#at !an be absorbe A Starch, a !arbo#y rate, is i;geste into isa!!#ari es an %onosa!!#ari es by t#e a!tion of s(e!ifi! en$y%es !alle amylasesA Li(i s are %a!ro%ole!ules t#at are !ataboli$e to glycerol an fatty acids by t#e en$y%e lipaseA Proteases igest (roteins into peptides an amino acidsA Peptidases igest peptides into a%ino a!i sA 3onosa!!#ari es, gly!erol an fatty a!i s, an a%ino a!i s are s%all enoug# to be absorbe a!ross t#e /all of t#e gastrointestinal tra!t an are use by !ells for buil ing ne/ %a!ro%ole!ules or to (roBi e energy (*0))A

Biology 257 LAB #11 !ontinue

)age 0#ree

Act $ t, #0: 'es!ri(tion of t#e !#e%i!al !o%(osition of .arbo#y rates, Li(i s, an )roteins
*ll carbohydrates !ontain t#e ele%ents !arbon, #y rogen, an o2ygen, t#e last t/o of /#i!# are (resent in t#e sa%e ratio as in /ater (2"7)A Carbohydrates %ay be !lassifie as mono , di , or polysaccharidesA 0#e mono an disaccharides rese%ble ea!# ot#er !onsi erably (are s/eet to t#e taste, for% a /#ite, granular substan!e, an are Bery soluble in /ater), but polysaccharides bear little rese%blan!e to t#e ot#er t/o !lassesA

Act $ t, #1: .#e%i!al 0ests of .arbo#y rates, )roteins, an Li(i s

Da!# of t#e Lab 0ables /ill be assigne one of t#e t#ree sets of testsH #o/eBer, you /ill be res(onsible for 1no/ing #o/ *LL of t#e tests are one, /#at t#e substrate is, /#at t#e en$y%e(s) are (/#ere a((li!able), /#et#er t#e test is (ositiBe or negatiBe for !ertain (ro u!tsA :ere are t#e t#ree sets of tests"

I2

C#r'oh,&r#te%:
7A Test for Starch and Starch Hydrolysis 0#is is a CualitatiBe test for lo!ali$ing star!# %ole!ulesA Proce&)re: 7) 6irst, #aBe one stu ent !olle!t 8s(it9 into one of t#e s%all, 25 %l bea1ersA ( 0#is 8Bolunteer9 /ill be oing t#is /#ile t#e ot#ers in t#e grou( are (ro!ee ing /it# t#e lab (roto!olA) 2) Into ea!# of t/o test tubes, (la!e 5 %l of t#e (re(are t#in star!# (asteA +) @sing a grease (en!il, nu%ber t#e tubes Tube ! an Tube " 4) 0o Tube !, a one ro( of Lugol5s io ine solution 5) =ote any !olor !#angeA I) 0o Tube ", a 4 5 ro(s of saliBa (as !olle!te fro% t#e 8Bolunteer9) ,) Set Tube " in a +,-. /ater bat# for J #our or %oreA 8) 4e%oBe Tube " fro% /ater bat# an test its !ontents by a ing one ro( of Lugol5s io ineA K) Is t#ere a !olor !#ange /#en t#e io ine solution is a e & 70) D2(lain t#e resultsA 2A #enedict$s Test 0#is is a CualitatiBe test s(e!ifi! for lo!ali$ing reducing sugars, /#i!# in!lu e all %onosa!!#ari es glu!ose, fru!tose, an gala!toseH an also so%e isa!!#ari es ; %annose, %altose, an la!toseA Proce&)re: 7) )la!e 5 %l of #enedict$s reagent into a test tubeH label t#is Tube !A 2) 0o Tube !, a four to fiBe ro(s of 34 "l)co%e solutionA +) )la!e Tube ! in a bea1er /ater;bat# oBer a Bunsen burnerH boil for 2 %inutesA 4) 4e%oBe Tube ! an let !ool slo/ly in a test tube ra!1A 5) W#ile !ooling, if t#ere is any !olor !#ange, or (re!i(itate for%ation, t#is in i!ates a (ositiBe rea!tionA I) 3a1e a note of any !olor !#ange (/#at !olor is it&), or if t#ere is any (re!i(itate for%ationA ,) )la!e 5 %l of Bene i!t5s solution into anot#er test tubeH label t#is Tube "A 8) 0o Tube ", a four to fiBe ro(s of 34 %)cro%e solutionA K) )la!e Tube " in a bea1er /ater;bat# oBer a Bunsen burnerH boil for 2 %inutesA 70) 4e%oBe Tube " an let !ool slo/ly in a test tube ra!1A 77) W#ile !ooling, if t#ere is any !olor !#ange, or (re!i(itate for%ation, t#is in i!ates a (ositiBe rea!tionA 72) 3a1e a note of any !olor !#ange (again, /#at !olor&), or if t#ere is any (re!i(itate for%ationA

Biology 257 LAB #11 !ontinue +A #arfoed$s Test 0#is is a CualitatiBe test s(e!ifi! for lo!ali$ing %onosa!!#ari es onlyA

)age 6our

Proce&)re: 7) )la!e 5 %l of #arfoed$s reagent in a test tubeA 2) * fiBe to si2 ro(s of 7o< glu!ose solutionA +) )la!e in a bea1er /ater;bat# oBer a Bunsen burnerH boil for 7 %inuteA 4) 4e%oBe tube fro% /ater bat# an set asi e in a test;tube ra!1 for 75 20 %inutes to !oolA 5) W#ile !ooling, /at!# for any !olor !#ange, or (re!i(itate for%ationA If eit#er or bot# of t#ese o!!ur, it in i!ates a (ositiBe rea!tionA I) 3a1e a note of any !olor !#angeL(re!i(itate for%ationA 4A Seli%anoff$s Test 0#is is a CualitatiBe test s(e!ifi! for istinguis#ing fru!tose fro% glu!ose an lo!ali$ing fru!tose onlyA Proce&)re: 7) 4un t#is test in u(li!ate, using t/o tubes /#i!# you (re;label Tube ! an Tube "A 2) Into ea!# of Tubes ! an ", (la!e 5 %l of Seli%anoff$s reagentA +) Into Tube !, a fiBe ro(s of 5< glu!ose solutionA 4) Into Tube ", a fiBe ro(s of 5< fru!tose solutionA 5) )la!e bot# Tubes ! an " into a bea1er /ater;bat# oBer a Bunsen burnerH boil for 7 %inuteA I) =ote any !olor !#ange (a !olor !#ange in i!ates a (ositiBe rea!tionA) W#at !olor is it& 5A &nversion of Sucrose Sin!e t#e isa!!#ari e su!rose, is !o%(ose of one glu!ose %ole!ule an one fru!tose %ole!ule !oBa; lently bon e to ea!# ot#er Bia a e#y ration synt#esis rea!tionH t#en, Bia a #y rolysis rea!tion, su!rose !an be e!o%(ose to its original t/o %onosa!!#ari e %ono%ersA 0#is test (roBi es t#e %eans by /#i!# su!# a #y rolysis rea!tion ta1es (la!eA Proce&)re: 7) * 5 %l of '( sucrose solution to ea!# of t/o test tubesH label t#ese Tube ! an Tube ". 2) 0o Tube !, a 5 !! of Bene i!t5s reagent an bring to a boil in t#e bea1er /ater bat#A +) 4e%oBe Tube ! fro% #eat i%%e iatelyA 4) 0o Tube ", a t/o ro(s of ilute (73) :.l an boil in t#e bea1er /ater bat# for 7 %inuteA 5) 4e%oBe Tube " fro% #eat an neutrali$e /it# an eCual a%ount an strengt# of =a>:A (D2(lain /#y t#is is i%(ortant to oA) I) 0est t#e !ontents of Tube " /it# 5 !! of #enedict$s reagentA ,) D2(lain t#e ifferent results for t#ese t/o 0ubes L testsA

II2

L p &%:

6ats are t#e esters (organi! salts) for%e by t#e union of a fatty a!i an an al!o#olA 0#e t#ree !o%%on fats in our foo s an in t#e bo y are olei!, (al%iti!, an steari!, /#i!# are for%e , res(e!tiBely, fro% olei!, (al%iti!, an steari! a!i s unite !#e%i!ally /it# t#e al!o#ol gly!erol (gly!erin)A >ne %ole!ule of gly!erol !an unite /it# t#ree fatty a!i s Bia a e#y ration synt#esis rea!tion as follo/s" 7A )igestion of Emulsified *at 0#is test !leBerly uses a (: in i!ator substan!e an an in ire!t %et#o of s#o/ing t#at li(i (fat) #as been !#e%i!ally bro1en o/nA

Biology 257 LAB #11 !ontinue

)age 6iBe

Proce&)re: 7) )our 70 %l of s/eet !rea% into a !lean test tubeH label it Tube !A 2) * 70 ro(s of 7< blue litmus solution to t#e !rea% to i%(art a lig#t blue !olorA +) 3i2 /ell /it# a /oo en stirring sti!1A 4) 'is(ense #alf of t#is %i2ture (about 5 %l) into anot#er test tube an label it Tube "A 5) * + %l of 5< Pancreatin solution to Tube "A I) * + %l of 0A5< +aHC,- to Tube !A ,) )la!e bot# tubes in t#e +,-. /ater bat# for one #ourA 8) 4e%oBe bot# 0ubes fro% t#e /ater bat#A 'o you obserBe any !olor !#ange in eit#er tube& K) D2(lain t#e resultsA@se a schematic representation of t#e !#e%i!al ste(s t#at #aBe o!!urre A

III2 Prote !%:

7A #iuret .eaction 0#e #iuret reaction is s(e!ifi! for !o%(oun s !ontaining t/o or %ore (e(ti e bon sA Proce&)re: 7) 0o + %l of 7< albu%in solution (egg /#ite), a + %l of 70< =a>: an one ro( of 7< .uS>4A 2) 3i2A +) * a itional ro(s of .uS>4 until a Biolet !olor is obtaine , in i!ating a (ositiBe rea!tionA 2A +inhydrin .eaction 0#e +inhydrin reaction is a test for al(#a a%ino a!i sA Proce&)re: 7) 0o 5 %l of 0A7< al!o#ol solution of gly!ine (or alanine), a 0A5 %l of 0A7< fres#ly (re(are =in#y rinA 2) :eat in a /ater bat#A =>0D !olor for%ationA +) 4e(eat ste(s 7 M 2 using + !! of 7< albu%in solution instea of an a%ino a!i solutionA 4) :eat to boiling t#e albu%in to /#i!# =in#y rin /as a e A 5) *llo/ to !oolA (W#at is t#e !olor&NNNNNNNNNNNNNNNNN) +A Heat Coagulation 0#is treat%ent e%onstrates t#e irreBersible enaturing effe!t on (rotein of e2(osure to #ig# #eatA Proce&)re: 7) )la!e 5 %l of un ilute albu%in (egg /#ite) in a test tube an (la!e in a bea1er /ater;bat# oBer a Bunsen burnerA 2) BoilA +) 'es!ribe t#e !#ange you obserBeA 4A )igestion of Protein 0#is treat%ent si%ulates one of t#e !#e%i!al igestion rea!tions in t#e bo y for (roteinA Proce&)re: 7) )la!e 7 2 !#un1s of !#o((e !oo1e egg /#ite fro% t#e :eat .oagulation e2er!ise aboBe into ea!# of t/o test tubesA 2) @sing a grease (en!il, nu%ber t#e tubes Tube ! an Tube "A +) Wit# a /oo en stirring sti!1, brea1 t#e egg;/#ite !#un1 in ea!# tube into so%e/#at s%aller (ie!esH but o not 8%as#9 entirely) 4) 0o Tube !, a 70 %l istille /ater (buffere to (: 8)A 5) 0o Tube ", a 70 %l of 5< )an!reatin solutionA I) )la!e bot# tubes in t#e +,-. /ater bat# for 7 J ; 2 #oursA ,) >bserBeH t#en es!ribe your resultsA

Biology 257 LAB #11 !ontinue

)age Si2

Act $ t, #5: *nalysis of t#e 4esults of .#e%i!al 0ests

0#ese are all colorimetric tests tests t#at !#ange !olor to in i!ate so%e !#e%i!al !#ange #as ta1en (la!eA (0#ey are not CuantitatiBe tests, but CualitatiBe onlyA)