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5 .

1R e v e r s iB n d i nogf a P r o t e ti 0
b li e ti.'l
n a L i g a n d : 0 x y g e n - B iPnrdoitnegi n s

Lake Powell,Arizona,August 2000.A family was vacation- 6,000to 30,000ppm under the swim deck, and atmos-
rng in a rented houseboat.They tumed on the electrical pheric 02 levels under the deck declined from 2lo/oto
generatorto power an air conditionerand a television. I2o/o.Even above the swim deck, CO levels of up to
About 15 minutes later, two brothers, aged 8 and 11, 7,200ppm were detected, high enoughto causedeath
jumped off the swrmdeck at the stem. Situatedimmedi- within a few minutes.
atelybelowthe deckwasthe exhaustport for the generator. How is a human affected by COFIb?At levels of less
Within two minutes,both boys were overcomeby the car- than 10% of total hemo$obin, sSrmptomsare rarely ob-
bon monoxide in the exhaust,which had becomeconcen- served.At I5o/o,theindividualexperiences mild headaches.
trated in the spaceunder the deck.Both drowned.These At 20o/oto 30o/o,the headacheis severeand is generallyac-
deaths,alongwith a seriesof deathsin the 1990sthat were companiedby nausea,dizziness,confusion,disorientation,
linked to houseboatsof similar design,eventuallyled to the and somevisual disturbances;these symptomsaxegener-
recall and redesignof the generatorexhaustassembly. ally reversedif the individual is treated with o>rygen.At
Carbonmonoxide(CO),a colorless,odorlessgas,is re- COIIblevelsof 30%to 5\o/o,theneurologicalsymptomsbe-
sponsiblefor more than half of yearly deathsdue to poison- come more severe,and at levels near 50%, the individual
ing worldwide. CO has an approximately250-fold greater losesconsciousness and can sink into coma.Respiratory
affinity for hemo$obin than does o4ygen. Consequently, failure may follow.With prolongedexposure,somedamage
relatively low levels of CO can have substantialand tragic becomespermanent.Death normally occurs when COFIb
effects.WhenCO combineswith hemo$obin,the complex levels rise above 60%0.Autopsy on the boys who died at
is referred to as carboxyhemoglobin, or COlIb. LakePowellrevealedCOIIblevelsof 59o/o and\2o/o'
SomeCO is produced by natural processes,but lo- Binding of CO to hemoglobin is affected by many
cally high levels generally result only from human activi- factors,including exercise(Fig. i) and changesin air
ties. Engineand furnaceexhaustsare important sources, pressure related to altitude. Becauseof their higher
as COis abyproduct of the ncomplete combustionof fos- base levels of COHb,smokersexposedto a sourceof
sil fuels. In the United Statesalone,nearly 4,000people CO often developsymptomsfaster than nonsmokers.
succumbto CO poisoning each year, both accidentally Individuals with heart, lung, or blood diseasesthat re-
and ntentionally. Many of the accidentaldeathsinvolve duce the availability of oxygen to tissues may also ex-
undetectedCObuildup in enclosedspaces,suchaswhen perience symptoms at lower Ievels of CO exposure.
a householdfurnace malfunctionsor leaks,venting CO Fetuses are at particular risk for CO poisoning,be-
into a home. However,CO poisoningcan also occur in causefetal hemoglobinhas a somewhathigher affinity
open spaces,as unsuspectingpeopleat work or play in- for CO than adult hemoglobin.Casesof CO exposure
hale the exhaustfrom generators,outboardmotors,trac- have been recorded in which the fetus died but the
tor engines,recreationalvehicles,or lau,nmowers. mother recovered.
Carbonmonoxidelevelsin the atmosphereare rarely (conti'rrued on nent PaSe)
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- gto
sites is 50 ppm for people working an eight-hour shift.
The tight binding of COto hemoglobinmeansthat COHb 8a
E
can accumulate over time as people are exposed to a
constantlow-levelsourceof CO. pb

In an average,healthy individual, 1% or less of the ()4


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 0
'o
20 40 60 80 1oo
Ievelsequilibrateat 50% in peoplewho breathe air con-
Carbon monoxide (PPm)
taining 570 ppm of CO for severalhours. Reliablemeth-
ods have been developedthat relate CO content in the FIGURE I Relationship betweenlevelsof COHb in blood and concen-
atmosphereto COHblevelsin the blood (F€ 1). In tests trationof CO in the surroundingair. Fourdifferentconditionsof expo-
ofhouseboatswith a generatorexhaustlike the one re- sure are shown, comparing the effects of short versusextended
sponsiblefor the Lake Powell deaths,CO levelsreached 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


hlernoglobin H- and(0t
Transports
Figure 5-14.
In addition to carryingnearly all the oxygenrequired by
for cells from the lungs to the tissues,hemoglobincarries
TwoModels
Suggest
Mechanisms
two end products of cellularrespiration-H+ and COz-
Binding
[ooperative from the tissuesto the Iungs and the kidneys,where
Biochemistsnow know a great deal about the T and R they are excreted.The CO2,producedby oxidationof
statesof hemoglobin,but much remainsto be learned organicfuels in mitochondria,is hydrated to form bicar-
about how the T -+ R transition occurs.T\.vomodelsfor bonate:
the cooperativebinding of ligandsto proteins with mul- CO2+ H2Oi- H* + HCOt
tiple binding sites have greatly influenced thinking
aboutthis problem. This reaction is catalyzedby carbonic anhydrase, an
The flrst model was proposedby JacquesMonod, enzyrneparticularly abundant in erythrocytes. Carbon
JeffriesW;rman,and Jean-PierreChangeuxin 1965,and dioxideis not very solublein aqueoussolution,and bub-
is called the MWC model or the concerted model blesof CO2wouldform in the tissuesandbloodif it were
(Fig. 5-15a). The concertedmodel assumesthat the 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+ o o.s
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
0246810
affinity of hemoglobinfor oxygendecreasesas H+ and pO2 ftPa)
CO2are bound,and 02 is releasedto the tissues.Con-
versely, in the capillaries of the lung, as CO2 is ex- FIGURE 5-16 Effectof pH on oxygenbindingto hemoglobin.ThepH
creted and the blood pH consequentlyrises, the 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 -
affinity of hemoglobinfor oxygen increasesand the surements on hemoglobinbindingare oftenperformedat pH 7.4.
protein binds more 02 for transport to the peripheral
tissues.This effect of pH and CO2 concentrationon rises, protonation of His HC3 promotes release of
the binding and release of oxygen by hemoglobinis oxygen by favoring a transition to the T state. proto-
calledthe Bohr effect, after ChristianBohr, the Dan- nation of the amino-terminalresidues of the a sub-
ish physiologist (and father of physicist Niels Bohr) units, certain other His residues,and perhaps other
groups has a similar effect.
who discoveredit in 1904.
The binding equilibrium for hemoglobinand one Thus we seethat the four polypeptide chainsof he-
molecule of oxygen can be designatedby the reaction moglobin communicatewith each other not only about
02 binding to their hemegroupsbut alsoabout H+ brnd-
Hb + 02 -+ HbO2 ing to specificamino acid residues.And there is still
but this is not a completestatement.To accountfor the more to the story.Hemoglobinalsobinds CO2,againin a
effect of H+ concentrationon this binding equilibrium, manner inverselyrelated to the binding of oxygen.Car-
we rewrite the reaction as bon dioxide binds as a carbamategroup to the a-amino
group at the amino-terminalend of each globin chain,
HHb*+Oz#HbOr+H+ forming carbaminohemoglobin:
where HlIb+ denotesa protonatedform of hemoglobin.
This equationtells us that the O2-saturationcurveof hemo- o
$obin is influencedby the H* concentration(Fig. b-f 6). TT- Oi TT
C+ H2N-C-C- --l--) C-N-C-C-
Both 02 and H* are boundby hemoglobin,but with inverse ltttl
afnniff. When the oxygen concentrationis high, as in the o RO o Ro
lungs, hemo$obin binds 02 and releasesprotons.When Amino-terminal Carbamino-terminal
the oxygen concentrationis low; as in the peripheral tis- residue residue
sues,H* is boundand 02 is released.
Oxygenand H+ are not bound at the samesites in This reaction produces H+, contributing to the Bohr ef-
hemoglobin.Oxygen binds to the iron atoms of the fect. The bound carbamates also form additional salt
hemes,whereasH* binds to any of severalamino acid bridges (not shown in Frg 5-9) that help to stabilize the
residuesin the protein. A major contribution to the T state and promote the release of oxygen.
Bohr effect is made by Hisra6(His HC3) of the B sub- When the concentration of carbon dioxide is high,
units. When protonated,this residueforms one of the as in peripheral tissues, some CO2 binds to hemoglobin
ion pairs-to Aspea(Asp FGI)-that helps stabilize and the affinity for 02 decreases, causing its release.
deoxyhemoglobinin the T state (Fig. 5-9). The ion Conversely, when hemoglobin reaches the lungs, the
pair stabilizesthe protonated form of His HCB,giving high oxygen concentration promotes binding of 02 and
this residue an abnormally high pK, in the T state. release of CO2. It is the capacity to communicate ligand-
The pK. falls to its normal value of 6.0 in the R state binding information from one potypeptide subunit to the
becausethe ion pair cannot form, and this residue is others that makes the hemoglobin molecule so beauti-
largely unprotonatedin oxyhemoglobinat pH 2.6, the fully adapted to integrating the transport of 02, CO2,
blood pH in the lungs. As the concentrationof H+ .--+.
and .t-l by en'throcytes.