Experiment 14: Proteins and Denaturing Agents

(adapted with permission of Dr. Ann Willbrand, !"#Ai$en%

Introduction
Proteins are pol&mers of amino a'ids. A t&pi'al protein ma& be 'omposed of
hundreds of amino a'ids. (he side 'hains of the amino a'id residues ma& 'ontain
nonpolar, neutral polar, a'idi', or basi' groups. (he primar& stru'ture of a protein is
determined b& the se)uen'e of amino a'ids, but the se'ondar& and tertiar& stru'tures of
proteins define their natural or nati*e states, whi'h are often folded. (his is 'alled the
nati*e 'onformation and is usuall& the state in whi'h the protein is most a'ti*e and
fun'tional. Proteins are held in their nati*e 'onformations b& a 'ombination of for'es:
h&drogen bonds, salt bridges (also 'alled ioni' intera'tions%, disulfide bridges, and
h&drophobi' intera'tions.
"hanging the 'onformation of a protein either temporaril& or permanentl& b&
disrupting these for'es is 'alled denaturation. Denaturation results in a loss of a'ti*it&.
!in'e the nati*e 'onformation is usuall& the most water soluble, disrupting the se'ondar&
and tertiar& stru'tures 'auses 'hanges in solubilit& and fre)uentl& results in pre'ipitation
of the protein from solution. +eagents or 'onditions that 'an 'ause denaturation are
'alled denaturing agents, these in'lude heat, p- 'hanges, al'ohol, hea*& metal salts, and
anions su'h as pi'rate and tannate.
-eat 'an suppl& $ineti' energ& to protein mole'ules, 'ausing their atoms to
*ibrate more rapidl&. (his will disrupt relati*el& wea$ for'es su'h as h&drogen bonds
and h&drophobi' intera'tions. (he most 'ommon example is obser*ed in 'oo$ing an egg.
-eat is also used in sterili.ation to denature and hen'e destro& the en.&mes in ba'teria.
Extremes of p- 'an 'ause a protein to denature. Although the ba'$bone of a
protein 'hain is neutral, the amino a'id residues that 'omprise the protein often 'ontain
a'idi' and basi' groups. (hese groups are usuall& 'harged and 'an form salt bridges with
a group of opposite 'harge. Extremes of p- 'an 'hange the 'harges on these a'idi' and
basi' groups, disrupting salt bridges.
/ess drasti' 'hanges in p- 'an also affe't the a'ti*it& and solubilit& of a protein.
/i$e indi*idual amino a'ids, proteins ha*e an isoele'tri' point at whi'h the number of
negati*e 'harges e)uals the number of positi*e 'harges. (his is fre)uentl& the point of
minimum water solubilit&. At the isoele'tri' p-, there is no net 'harge on the mole'ule.
0ndi*idual mole'ules ha*e a tenden'& to approa'h one another, 'oagulate, and pre'ipitate
N
C O
H C O
N H
h&drogen bonds
NH
2
11"
salt bridge
S S
disulfide bridge
Ar
Ar
-&drophobi'
0ntera'tion
out of solution. At a p- abo*e or below the isoele'tri' p-, the mole'ules ha*e a net
negati*e or positi*e 'harge, respe'ti*el&. (hus when protein mole'ules approa'h ea'h
other, the& ha*e the same o*erall 'harge and repulse ea'h other. (his pre*ents
'oales'en'e and pre'ipitation. "asein, the ma2or protein in mil$, pro*ides a good
example. At its isoele'tri' p- of 4.3 it will pre'ipitate, but it is soluble at the normal p-
of mil$, 3.3. When mil$ sours, la'ti' a'id is produ'ed that lowers the p-. "asein
pre'ipitates, forming white 'urds also $nown as 'ottage 'heese.
+eagents su'h as ethanol that are 'apable of forming intermole'ular h&drogen
bonds with protein mole'ules will disrupt the intramole'ular h&drogen bonding within
the mole'ule. A 456 solution of al'ohol 'an be used as a disinfe'tant, be'ause the
al'ohol fun'tions to denature the proteins in ba'teria. A 456 solution is used be'ause it
will effe'ti*el& penetrate the ba'terial 'ell wall, a 786 solution 'oagulates proteins at the
surfa'e of the 'ell wall, forming a 'rust that pre*ents the al'ohol from penetrating into the
'ell.
!alts of metal ions su'h as mer'ur&(00%, lead(00%, and sil*er 'an form strong bonds
with disulfide groups and with the 'arbox&late ions of the a'idi' amino a'ids. (hus, the&
disrupt both disulfide bridges and salt lin$ages and 'ause the protein to pre'ipitate out of
solution as an insoluble metal#protein salt. (his propert& ma$es some of the hea*& metal
salts suitable for use as topi'al antisepti's. -owe*er, most hea*& metal salts are toxi'
when ta$en internall&, be'ause the& pre'ipitate the proteins of all the 'ells with whi'h
the& 'ome into 'onta't. !ubstan'es high in protein, su'h as egg whites and mil$, are used
as antidotes for hea*& metal poisoning, be'ause their proteins readil& 'ombine with the
metal ions to form insoluble solids. (he resulting insoluble matter must immediatel& be
remo*ed from the stoma'h b& the use of an emeti' to pre*ent the gastri' 2ui'es from
destro&ing the protein and on'e again liberating the poisonous hea*& metal ions.
Pi'rate and tannate ions are the 'on2ugate bases of pi'ri' and tanni' a'ids,
respe'ti*el&. (hese negati*el& 'harged ions 'ombine with the basi' amino a'ids, whi'h
are positi*el& 'harged. (his disrupts intramole'ular salt bridges. 0n the manufa'ture of
leather, tanni' a'id is used to pre'ipitate the proteins in animal hides. (his is 'ommonl&
$nown as the tanning pro'ess. (anni' a'id, whi'h is found in tea, is sometimes used to
treat burns. (he a'id 'ombines with the protein in the exposed areas to form a leather&
'oating that ex'ludes air and stops the loss of bodil& fluids.
0n this exer'ise, the effe't of se*eral denaturing agents on two different proteins
will be studied. Albumin is a simple globular protein. 0t is soluble in water and dilute
salt solutions su'h as isotoni' saline (5.76 9a"l%. "asein is the ma2or protein 'omponent
of mil$. 0ts solubilit& is *er& sensiti*e to p-.
Laboratory Activities
A. Examine the effe't of heat on the solubilit& of albumin
:. Examine the effe't of p- 'hanges on the solubilit& of albumin and 'asein
". Examine the effe'ts of 786 ethanol, lead(00% nitrate, sil*er nitrate, and tanni' a'id on
albumin and 'asein
Procedure
A. The effect of heat
Pla'e about 1 m/ of ;6 albumin in a test tube and heat it in a hot water bath for a
few minutes. "ompare the appearan'e to the albumin solution at room temperature.
B. The effect of pH changes
Pla'e about 1 m/ of ;6 albumin in ea'h of four test tubes. Add < m/ of water to
the first. (his tube will ser*e as a 'ontrol. (o the se'ond, add 156 9a1- dropwise until
the p- is 14. ((o do this, add a 'ouple of drops of 9a1- to the tube, stir thoroughl& with
a stirring rod, then tou'h the stirring rod to a pie'e of p- paper to 'he'$ &our p-.% (o
the third, add 5.86 sodium bi'arbonate solution to p- 7, and to the fourth, add ;6 -"l
to p- ;. +e'ord &our obser*ations on the data sheet.
+epeat the abo*e tests using ;6 'asein solution. +e'ord &our obser*ations.
"ompare the results for the two tests.
C. The effects of ethanol, lead(II nitrate, silver nitrate, and tannic acid
Pla'e 1 m/ portions of ;6 albumin in ea'h of 8 test tubes. (he first tube is &our
'ontrol. (o the se'ond tube, add 1 m/ of 786 ethanol, to the third, add se*eral drops of
lead(00% nitrate, to the fourth, add se*eral drops of sil*er nitrate, and to the last, add
se*eral drops of tanni' a'id. "ompare ea'h of the test solutions to the 'ontrol. +e'ord
&our obser*ations.
+epeat the abo*e tests, substituting ;6 'asein solution for the albumin. +e'ord
&our obser*ations. Are &our results 'onsistent with the results from albumin=
!"peri#ent $% 9ame:>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Laboratory &ecord Date:>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
A. -eat effe't
+e'ord &our obser*ations:
:. p- Effe'ts
+e'ord &our obser*ations in the 'hart:
'ediu# (pH !ffect on Albu#in !ffect on Casein
Water (p- 4% ('ontrol%
15 6 9a1- (p- 14%
8 6 9a-"1
<
(p- 7%
;6 -"l (p- ;%
". Effe'ts of ethanol, lead(00% nitrate, sil*er nitrate, and tanni' a'id
+e'ord &our obser*ations in the 'hart:
Agent !ffect on Albu#in !ffect on Casein
9one ("ontrol%
78 6 ethanol
Pb
;?
Ag
?
(anni' a'id
@uestions:
1. 0n &our own words, explain the effe't of heat on the solubilit& of albumin.
;. 0s 'asein more soluble in base or in a'id= Wh&=
<. When mil$ sours, la'ti' a'id is produ'ed and a white pre'ipitate forms. What is this
pre'ipitate= Explain what is happening.
4. Did &ou obser*e an& differen'es in the solubilities of 'asein and albumin at p- ;=
at p- 7=
at p- 14=
Arom &our obser*ations, whi'h is more affe'ted b& p-, 'asein or albumin=
4. What $inds of disruptions to the nati*e 'onformation o''ur in ea'h of the following=
0n other words, are h&drogen bonds, disulfide lin$ages, et'. disrupted=
Albumin is heated.
Al'ohol is added to 'asein.
Betal ions are added to 'asein.
(anni' a'id is added to albumin.