5 .
1R e v e r s iB n d i nogf a P r o t e ti 0
Lake Powell,Arizona,August 2000.A family was vacation-
rng in a rented houseboat.They tumed on the electrical
generatorto power an air conditionerand a television.
About 15 minutes later, two brothers, aged 8 and 11,
jumped off the swrmdeck at the stem. Situatedimmedi-
atelybelowthe deckwasthe exhaustport for the generator.
Within two minutes,both boys were overcomeby the car-
bon monoxide in the exhaust,which had becomeconcen-
trated in the spaceunder the deck.Both drowned.These
deaths,alongwith a seriesof deathsin the 1990sthat were
linked to houseboatsof similar design,eventuallyled to the
recall and redesignof the generatorexhaustassembly.
Carbonmonoxide(CO),a colorless,odorlessgas,is re-
sponsiblefor more than half of yearly deathsdue to poison-
ing worldwide. CO has an approximately250-fold greater
affinity for hemo$obin than does o4ygen. Consequently,
relatively low levels of CO can have substantialand tragic
effects.WhenCO combineswith hemo$obin,the complex
is referred to as carboxyhemoglobin, or COlIb.
SomeCO is produced by natural processes,but lo-
cally high levels generally result only from human activi-
ties. Engineand furnaceexhaustsare important sources,
as COis abyproduct of the ncomplete combustionof fos-
sil fuels. In the United Statesalone,nearly 4,000people
succumbto CO poisoning each year, both accidentally
and ntentionally. Many of the accidentaldeathsinvolve
undetectedCObuildup in enclosedspaces,suchaswhen
a householdfurnace malfunctionsor leaks,venting CO
into a home. However,CO poisoningcan also occur in
open spaces,as unsuspectingpeopleat work or play in-
hale the exhaustfrom generators,outboardmotors,trac-
tor engines,recreationalvehicles,or lau,nmowers.
Carbonmonoxidelevelsin the atmosphereare rarely
dangerous,rangingfrom lessthan 0.05parts per miliion
(ppm) in remote and unintrabited areasto 3 to 4 ppm
in somecities of the northern hemisphere.In the United
States,the government-mandated(OccupationalSafety
and Health Administration,OSHA)Iimit for CO at work-
sites is 50 ppm for people working an eight-hour shift.
The tight binding of COto hemoglobinmeansthat COHb
can accumulate over time as people are exposed to a
constantlow-levelsourceof CO.
In an average,healthy individual, 1% or less of the
total hemoglobinis complexedas COHb.SinceCO is a
product of tobacco smoke, many smokershave COHb
levelsin the range of 3% to 8% of total hemoglobin,and
the levels can rise to l5o/ofor chain-smokers.COHb
Ievelsequilibrateat 50% in peoplewho breathe air con-
taining 570 ppm of CO for severalhours. Reliablemeth-
ods have been developedthat relate CO content in the
atmosphereto COHblevelsin the blood (F€ 1). In tests
ofhouseboatswith a generatorexhaustlike the one re-
sponsiblefor the Lake Powell deaths,CO levelsreached
b li e ti.'l
n a L i g a n d : 0 x y g e n - B iPnrdoitnegi n s
6,000to 30,000ppm under the swim deck, and atmos-
pheric 02 levels under the deck declined from 2lo/oto
I2o/o.Even above the swim deck, CO levels of up to
7,200ppm were detected, high enoughto causedeath
within a few minutes.
How is a human affected by COFIb?At levels of less
than 10% of total hemo$obin, sSrmptomsare rarely ob-
served.At I5o/o,theindividualexperiences mild headaches.
At 20o/oto 30o/o,the headacheis severeand is generallyac-
companiedby nausea,dizziness,confusion,disorientation,
and somevisual disturbances;these symptomsaxegener-
ally reversedif the individual is treated with o>rygen.At
COIIblevelsof 30%to 5\o/o,theneurologicalsymptomsbe-
come more severe,and at levels near 50%, the individual
losesconsciousness and can sink into coma.Respiratory
failure may follow.With prolongedexposure,somedamage
becomespermanent.Death normally occurs when COFIb
levels rise above 60%0.Autopsy on the boys who died at
LakePowellrevealedCOIIblevelsof 59o/o and\2o/o'
Binding of CO to hemoglobin is affected by many
factors,including exercise(Fig. i) and changesin air
pressure related to altitude. Becauseof their higher
base levels of COHb,smokersexposedto a sourceof
CO often developsymptomsfaster than nonsmokers.
Individuals with heart, lung, or blood diseasesthat re-
duce the availability of oxygen to tissues may also ex-
perience symptoms at lower Ievels of CO exposure.
Fetuses are at particular risk for CO poisoning,be-
causefetal hemoglobinhas a somewhathigher affinity
for CO than adult hemoglobin.Casesof CO exposure
have been recorded in which the fetus died but the
mother recovered.
(conti'rrued on nent PaSe)
gto
8a
E
pb
()4
0
'o
20 40 60 80 1oo
Carbon monoxide (PPm)
FIGURE I Relationship betweenlevelsof COHb in blood and concen-
trationof CO in the surroundingair. Fourdifferentconditionsof expo-
sure are shown, comparing the effects of short versusextended
exposure,and exposureat restversusexposureduring light exercise'
 164 ProteF
i nu n c t i o n
i ]

It may seem surprising that the loss of half of one,s

hemoglobin to COHb can prove fatal-we know that
people with any of several anemic conditions manage [o
function reasonably well with half the usual complement
of active hemoglobin. However, the binding of CO to he-
moglobin does more than remove protein from the pool
available to bind oxygen It also affects the affinity of the
remaining hemoglobin subunits for oxygen. As CO binds
to one or two subunits of a hemoglobin tetramer, the
affinity for 02 is increased substantially in the remaining
subunits (Fig 2) Thus, a hemoglobin tetramer wrth two
bound CO molecules can efficiently bind 02 in the
lungs-but it releases very little of it in the tissues.
Oxygen deprivation in the tissues rapidly becomes severe.
To add to the problem, the effects of CO are not limited
to interference with hemoglobin function. CO binds to
other heme proteins and a variety of metalloproteins. 48I2
The effects of these interactions are not yet well under- pO2(kPa)
stood, but they may be responsible for some of the FIGURE2 Several oxygen-binding
curves:for normalhemoglobin, he-
longer-term effects of acute but nonfatal CO poisoning. moglobinfrom an anemicindividualwith only 50% of her hemoglo-
When CO poisoning is suspected, rapid evacuation bin functional,and hemoglobin
from an individualwith 50% of his
of the person away from the CO source is essential,but hemoglobinsubunitscomplexedwith CO. The pO2 in humanlungs
this does not always result in rapid recovery. When an andtissuesis indicated
individual is moved from the CO-polluted site to a nor_
mal, outdoor atmosphere, 02 begins to replace the CO in of 3 atm (303kPa) is supplied.Thus,rapid treatmentby
hemoglobin-but the COHb levels drop only slowly. The a properly equippedmedicalteam is critical.
half-time is 2 to 6 5 hours, depending on individual and Carbonmonoxide detectorsin all homesare highly
environmental factors. If 100% oxygen is administered recommended.This is a simple and inexpensivemea-
with a mask, the rate of exchange can be increased sure to avoid possibletragedy.After completingthe re-
about fourfold; the half-time for O2-CO exchange can be search for this box, we immediately purchasedseveral
reduced to tens of minutes if 100% oxygen at a pressure new CO detectorsfor our homes.

The expressionfor 0 (seeEqn 5-8) is n11of greater than 1 indicates positive cooperativity in lig-
[L]" and binding. This is the situation observed in hemoglobin,
o_
lLl" + Ku 3 (5-14) in which the binding of one molecule of ligand facilitates
the binding of others. The theoretical upper limit for ns is
Rearranging,then taking the log of both sides, yields
reached when ns : ?L In this case the binding would be
e _ ILl" completely cooperative: all bindiry sites on the protein
(5-15)
r-0 Kd would bind ligand simultaneously, and no protein mole_
cules partially saturated with ligand wor:ld be present un-
h-(*) :nrogtlJ -rogKd
$ ts-rol der any conditions. This limit is never reached in practice,
and the measured value of ns is always less than the ac-
wnereKa: [L]6s.
tual number of ligand-binding sites in the protein.
Equation 5-16 is the Hill equation, and a plot of log
- Annll of less than I indicates negative cooperativ_
W/Q 0)l versus log [L] is called a Hill plot. Based on the ity, in which the binding of one molecule of ligand i,m-
equation, the Hill plot should have a slope of n However,
pedes the binding of others. Well-documented cases of
the experimentally determined slope actually reflects not
negative cooperativity are rare.
the number of binding sites but the degree of interaction
To adapt the Hill equation to the binding of oxygen
between them. The slope of a Hill plot is therefore de_
to hemoglobin we must again substitute pO2 for [L] and
noted by ns, the Hill coefficient, which is a measure of
P{o for Ka:
the degree of cooperativity. If nsequals 1, ligand binding
is not cooperative, a situation that can arise even in a mul_
tisubunit protein if the subunits do not communicate. An .-(*) : nbspoz - nlogp5s (b-17)
 5 . 1R e v e r s iB
b li e n a L i g a n d : 0 x y g e n - B iPnrdoitnegi n s["t]
n d i nogfa P r o t e ti o

subunitsof a cooperativelybinding protein are function-

ally identical,that eachsubrnit can exist in (at least) two
conformations,and that all subunitsundergothe transi-
Hemoglobin tion from one conformationto the other simultaneously.
high'a{Iinity In this model, no protein has individual subunits in dif-
1 state \,,
nu: | .u
ferent conformations. The two conformations are in
s
equilibrium.The hgandcan bind to either conformation,
l*0
but binds eachwith different affinity.Successivebinding
of ligand molecules to the low-affinity conformation
-1
l'');",'
Iow-affrnity
(which is more stablein the absenceof ligand) makesa
transition to the high-affinity conformationmore likely.
-2 In the second model, the sequential model
(Fig. 5-15b), proposedin 1966by DanielKoshlandand
colleagues,ligandbinding can induce a changeof con-
_J
-2 -1 formation in an individual subunit. A conformational
log p02 change in one subunit makes a similar change in an
adjacentsubunit, as well as the binding of a secondlig-
FIGURE 5-14 Hill plots for oxygenbindingto myoglobinand hemo-
-1,
and molecule,more likely. There are more potential
globin.When nr1: The maximum
thereis no evidentcooperativity. intermediatestatesin this model than in the concerted
degreeof cooperativity observedfor hemoglobincorresponds approxi- model. The two models are not mutually exclusive;the
matelyto ng : 3. Notethatwhilethisindicates a highlevelof cooper-
concertedmodelmaybe viewedasthe "all-or-none"lim-
ativity,ns is lessthann, the numberof O2-bindingsitesin hemoglobin.
iting caseof the sequentialmodel.In Chapter6 we use
Thisis normalfor a proteinthat exhibitsallostericbindingbehavior
thesemodelsto investigateallostericenzyrnes'

Hill plots for myoglobin and hemoglobin are given in Also

Figure 5-14.
for
TwoModels
Suggest
Mechanisms
Binding
[ooperative
Biochemistsnow know a great deal about the T and R
statesof hemoglobin,but much remainsto be learned
about how the T -+ R transition occurs.T\.vomodelsfor
the cooperativebinding of ligandsto proteins with mul-
tiple binding sites have greatly influenced thinking
aboutthis problem.
The flrst model was proposedby JacquesMonod,
JeffriesW;rman,and Jean-PierreChangeuxin 1965,and
is called the MWC model or the concerted model
(Fig. 5-15a). The concertedmodel assumesthat the
hlernoglobin H- and(0t
Transports
In addition to carryingnearly all the oxygenrequired by
cells from the lungs to the tissues,hemoglobincarries
two end products of cellularrespiration-H+ and COz-
from the tissuesto the Iungs and the kidneys,where
they are excreted.The CO2,producedby oxidationof
organicfuels in mitochondria,is hydrated to form bicar-
bonate:
CO2+ H2Oi- H* + HCOt
This reaction is catalyzedby carbonic anhydrase, an
enzyrneparticularly abundant in erythrocytes. Carbon
dioxideis not very solublein aqueoussolution,and bub-
blesof CO2wouldform in the tissuesandbloodif it were
not convertedto bicarbonate.As you can seefrom the

AllO A1lE
r-Y-)
XX
l=o-E-E-m ll
FIGURE 5-15 Two generalmodelsfor the intercon-
oo-oo-m-re=- versionof inactiveand activeforms of a proteindur-
1l 1l 1l\
lr\tvtv 11 1t 1t 1t ing cooperativeligandbinding'Althoughthe models
on ETI G(])-Im-trt-m-ul may be appliedto any protein-includingany en-
(J., ul OO=-OO.-@-I=-I I zyme(Chapter 6)-that exhibitscooperative
we show herefour subunitsbecausethe modelwas
binding,

1l 1l 1r 1r\11 1f 1t originallyproposedfor hemoglobin.(a) In the con-

6XD fl,lLl GG)-EO-m-m-ffi certed,or all-or-none,model(MWC model),all sub-
IY]
tTl oo-oo-er.-[I)=-ul unitsare postulated to be in the sameconformation,
1l 1l 1t 1t 1t\1t 1l eitherall O (low affinityor inactive)or all tr (high
6YD tilL] (n{) -nlD -tTlt -ttTil-EItl affinityor active).Depending K1,
on the equilibrium,
aJ trI 60:60:m-re-ETt betweenO and D forms,the bindingof one or more

1l 1l 1t 1t 1t 1r\1r ligandmolecules (L)will pull the equilibriumtoward

the ! form.Subunits with boundL areshaded.(b) ln
GXD lxlxt cYD .mD -Etil -tr|il-trit
6)0 m 66:66: m:EGj=- l-Lft the sequential model,eachindividualsubunitcan be
in eitherthe O or n form.A very largenumberol
(a) ft) is thusPossible.
conformations
 !"1 P r o t e Fi nu n c t i o n
reaction catalyzedbycarbonicanhydrase,the hydration
of CO2resultsin an increasein the H+ concentration(a
decreasein pH) in the tissues.The bindingof oxygenby
hemoglobinis profoundly influenced by pH and CO2
concentration,so the interconversionof CO2and bicar-
bonate is of great importance to the regulation of
oxygenbinding and releasein the blood.
Hemoglobintransports abottt 400/o of the total H+
and 15% to 20o/oof the CO2formed in the tissues [o
the lungs and kidneys.(The remainderof the H+ is ab-
sorbedby the plasma'sbicarbonatebuffer; the remain-
der of the CO2is transportedas dissolvedHCO| and
CO2.)The binding of H* and CO2is inverselyrelated
to the binding of oxygen.At the relativelyIow pH and
high CO2 concentrationof peripheral tissues, the
affinity of hemoglobinfor oxygendecreasesas H+ and
CO2are bound,and 02 is releasedto the tissues.Con-
versely, in the capillaries of the lung, as CO2 is ex-
creted and the blood pH consequentlyrises, the
affinity of hemoglobinfor oxygen increasesand the
protein binds more 02 for transport to the peripheral
tissues.This effect of pH and CO2 concentrationon
the binding and release of oxygen by hemoglobinis
calledthe Bohr effect, after ChristianBohr, the Dan-
ish physiologist (and father of physicist Niels Bohr)
who discoveredit in 1904.
The binding equilibrium for hemoglobinand one
molecule of oxygen can be designatedby the reaction
Hb + 02 -+ HbO2
but this is not a completestatement.To accountfor the
effect of H+ concentrationon this binding equilibrium,
we rewrite the reaction as
HHb*+Oz#HbOr+H+
where HlIb+ denotesa protonatedform of hemoglobin.
This equationtells us that the O2-saturationcurveof hemo-
$obin is influencedby the H* concentration(Fig. b-f 6).
Both 02 and H* are boundby hemoglobin,but with inverse
afnniff. When the oxygen concentrationis high, as in the
lungs, hemo$obin binds 02 and releasesprotons.When
the oxygen concentrationis low; as in the peripheral tis-
sues,H* is boundand 02 is released.
Oxygenand H+ are not bound at the samesites in
hemoglobin.Oxygen binds to the iron atoms of the
hemes,whereasH* binds to any of severalamino acid
residuesin the protein. A major contribution to the
Bohr effect is made by Hisra6(His HC3) of the B sub-
units. When protonated,this residueforms one of the
ion pairs-to Aspea(Asp FGI)-that helps stabilize
deoxyhemoglobinin the T state (Fig. 5-9). The ion
pair stabilizesthe protonated form of His HCB,giving
this residue an abnormally high pK, in the T state.
The pK. falls to its normal value of 6.0 in the R state
becausethe ion pair cannot form, and this residue is
largely unprotonatedin oxyhemoglobinat pH 2.6, the
blood pH in the lungs. As the concentrationof H+
o o.s
0246810
pO2 ftPa)
FIGURE 5-16 Effectof pH on oxygenbindingto hemoglobin.ThepH
o f b l o o di s 7 . 6 i n t h e l u n g sa n d 7 . 2i n t h e t i s s u e E
s .x p e r i m e n tmael a -
surements on hemoglobinbindingare oftenperformedat pH 7.4.
rises, protonation of His HC3 promotes release of
oxygen by favoring a transition to the T state. proto-
nation of the amino-terminalresidues of the a sub-
units, certain other His residues,and perhaps other
groups has a similar effect.
Thus we seethat the four polypeptide chainsof he-
moglobin communicatewith each other not only about
02 binding to their hemegroupsbut alsoabout H+ brnd-
ing to specificamino acid residues.And there is still
more to the story.Hemoglobinalsobinds CO2,againin a
manner inverselyrelated to the binding of oxygen.Car-
bon dioxide binds as a carbamategroup to the a-amino
group at the amino-terminalend of each globin chain,
forming carbaminohemoglobin:
o
TT- Oi TT
C+ H2N-C-C- --l--) C-N-C-C-
ltttl
o RO o Ro
Amino-terminal Carbamino-terminal
residue residue
This reaction produces H+, contributing to the Bohr ef-
fect. The bound carbamates also form additional salt
bridges (not shown in Frg 5-9) that help to stabilize the
T state and promote the release of oxygen.
When the concentration of carbon dioxide is high,
as in peripheral tissues, some CO2 binds to hemoglobin
and the affinity for 02 decreases, causing its release.
Conversely, when hemoglobin reaches the lungs, the
high oxygen concentration promotes binding of 02 and
release of CO2. It is the capacity to communicate ligand-
binding information from one potypeptide subunit to the
others that makes the hemoglobin molecule so beauti-
fully adapted to integrating the transport of 02, CO2,
.--+.
and .t-l by en'throcytes.