EGERTONUNIVERSI
TY
BMED31
8:BIOCHEMI
CALTECHNI
QUES
LECTURENOTES
I
n troduc
tion
Biochemist
r yi sthes t
u dyofc hemi calp rocessestha ttakep laceinl i
vingorga ni
smswi t
ht heult
ima te
aimo fu nder s
tan di
n gt hen atureo fl i
fei nmo l
eculart erms .Bi ochemicals t
u di
esa rea i
dedb y
avail
ab i
li
tyo fa n al
y t
icalt e
chn i
q uest ha ta dvancet hekn owledgeo ft hen a t
urea ndr e
lati
onships
betweenb i
o logi
ca lmo lecul
essu c
ha sn ucleicac i
dsa ndprotei
n sa ndtheircell
ularfu ncti
on.
I
d enti
ficat
io no fc onstitu
ents:e xp e
r imenta ldesi
gn
Exp eri
mentsa rec a refull
yd esign ed,e xecuteda ndd ataa nalyzedtoa nswe rs peci
ficq ues
tio
n so r
hyp o
theses.Ex perimen ta
ld e
signi nvolvesan umbe rofs t
age s:
·theidenti
fi cati
ono ft hes ubj
e c
tfo re xperime nt
ali nv
esti
ga ti
o n;
·thec ri
tic
a le v
alu a
tiono fthec urr ents ta
t eofkn owl e
dge( the‘ li
terature
’)oft hec hosensubje
cta rea
notingthes tr e
ngt hsa ndwe aknes seso ft heme t
ho dol
ogiesp reviousl
ya ppl
ieda ndthen ewhy pot
he ses
whiche mer gedfr omt hestudies
;
·thefo rmula ti
ono ft heq ues
tiono rhy pot
he sistob eaddressedb yt heplannedexperime n
t;
·thec are
fu ls e
lectiono ftheb iol
o gicals ystem( s
p eci
es,inv i
voo rinv i
tro)tob eusedfo rthestudy;
·theid e
ntificationo ft hevariablet ha tist ob estudie
d;t hec o n
siderat
ionoft heo therv a
riabl
est hat
willn ee
dt ob ec ontrolle
ds ot ha tt hes electedv aria
b l
ei st heo nlyfa ct
ort hatwi lld e
termin
et he
experimentalo utcome ;
·thed esi
gno ft hee xp er
imentin cludingt hes tatist
icalanalysiso fther esul
ts,c a
r efulevalu
ati
o no f
thema teri
a lsanda p paratustob eu seda ndt hec o
n sequ
entialpo tent
ialsafetyaspectso fthestudy;
·thee xecutiono ft hee xperi
me nti ncludinga pp
r opri
atec ali
b rati
onsa ndc o
ntrols
,wi thac arefully
wr i
ttenrecor doft heo ut
comes;
·ther epl
icationo ft hee xp
e r
imen ta sn ece
s saryfo rtheu namb iguousanalysi
so ftheo utcomes;
·t hee valua ti
ono ft heo utcome si nclud i
ngt hea p p
licati
o no fa ppr
op ri
ates tatis
tica
lt es
t st o
quantit
ativ
ed atawhe reappli
cab l
e ;
·thefo rmula ti
ono ft hema inconc lusi
o nstha tcanb edr a
wnfr o mther esu
lts;
·thefo rmula ti
ono fn ewhy pothes esa ndoffu turee xper
ime ntstha temergefr omt hes t
udy
Quant
it
a t
ivemeasu
rement
a)Uni
tsofme as
urement
TheFrenchSystè
meInter
nat
io
nalďUni
té
s(t
heSIs
yst
em)isthea
ccep
tedc
onve
nti
onforal
luni
tsof
meas
urement.Onemoleofasubs
tan
cei
sequ
alt
oit
smo l
ecul
armassex
pre
sse
dingrams,whe
rethe
1
,molecularma ssi sthes umo fthea tomicma sse
so ft hec onsti
t uen
ta t
oms.No tethatt het e
r m
molecularma ssisp referredtot heo ldertermmo lec
u l
arwe ight.TheSIu nitofc oncent
r at
ioni s
-3
expres
s e
di nt e
rmso fmo l
esp e
rc ubicme tre(molm )( Table1 )
.I np rac
ticethisisfa rtoolargefo r
3 -
3
normallabora t
oryp urpos
esa n
dau nitb ase
do nacu b
icd eci
me ter(dm,1 0 m)i spreferred.
Themo lari
tyo fas o
lutio
no fas u
b st
anc eexpres
sesthen u mb e
ro fmo l
esoft hesubstanceinonec u
b ic
deci
met e
ro fs ol
ution
.I ti
se xpr
essedb ythes ymbolM.
I
ts houldben otedthata t
omicandmo lecularma s
sesa rebo thexp re
s s
edinDa lton
s( Da)orkiloDa lt
on s
(kDa),whe reo neDa l
tonisa natomicma ssu ni
tequalt oon etwe l
ft hoft hema sso fonea t
omo ft he
1
2
Ci sotope
.Ho we v
er,b io
chemist
sp refert ouset het er mr elativ
emo le
cularma ss( Mr).Thi si s
1
2
defin
eda st hemo lecul
arma ssofas ubstancerelat
ivet oo ne-t
we lftho fthea tomicma ssoft he C
i
s o
tope.Mrt hereforehasn ouni
ts.
Thust herelativ
emo lecula
rma sso fsodiumc hlo
rideis23( Na)p lus35. 5(Cl)i.e.58. 5,sot haton e
moleis58. 5gr ams.I fthiswasdissolvedinwa t
eran da d
ju s
t e
dt oat ota
lv ol
umeo f1d m3t hesolut
io n
wouldb eo n emo l
ar( 1M) .Bi o
logicals ubsta
ncesa r emo s
tfr equen
tlyfo unda tr el
ati
velyl ow
conce
ntrati
on sa ndini nv itr
omo de
ls y s
temst hev o
lume so fs tocks olut
ionsr egularl
yu sedfo r
experi
me nt
alp urposesarealsosmall.Thec onseque
ncei stha texperimentalsolu
tionsareu s
uallyinthe
mM,n Ma ndp Mr anger a
therthanmo lar.
Table1:SIu ni
ts-Basicuni
ts
Qu
ant
it
y SIu
nit Sy
mbo
l
Le
ngt
h Me
tre m
Ma
ss Ki
lo
gra
m kg
Ti
me s
eco
nds s
El
ect
ricc
urr
ent Amp
ere A
Te
mpe
rat
ure Ke
lvi
n K
Lu
min
ousi
nte
nsi
ty Ca
nde
la c
d
Amo
unto
fsu
bst
anc
e Mo
le mo
l
I
nscie
nceitiscri
ti
caltha
texper
iment
sandtheob
ser
vati
onsrec
orde
dar
erepea
tabl
e.
There
forequ a
nti
tat
iveobser
vati
onsaremu c
hmo r
ev a
luabl
et ha
nqual
it
ati
ve.Whenan ume
ric
al
val
ueisgi v
ent oac hara
cte
ris
ticital
lowsfordire
ctc o
mparis
onbe
tweenobs
erv
ati
onsmad
eb y
2
, di
fferentp eopleora tdifferentti
mes .
Qu a
nti
tativeme asuremen tiscoll
ecti
ngi nformationthatisnumericinn a
ture.
Qu a
nti
tatived ataha sv al
ueso fav ariabledifferi
ngi na mountinn ume r
ict ermsr a
therthanin
desc
ript
ivet erms.
Qu a
nti
tativeme t
hodsa rer es
earchte chniquesthatareu se
dt ogatherq ua
n t
itat
ived a
ta-in
formati
on
deal
ingwi thn umbersa nda nyt
hingtha tisme asurable
.St at
isti
cs,tablesandgr aphs,areofte
nu s
ed
topresentt her esul
t soft heseme thod s
.The ya r
et here
foret ob ed i
st
inguishedfromq ual
it
ativ
e
methods
.
c)Accuracy&Pr ecision
Accurac
ya n dp reci
sions houldb ec o nsi
deredwhe therc oll
ect
ingd at
ao rme asuri
ng mater
ials
.
Accuracyr eferstoho wc lo
seame asure dvalueistothet r
uev al
ue.
Preci
sio
nr eferstoho wr eproduc
ibleame a
surementis.Ify our epeatame asurementan umberof
ti
mes,itisho wc losethen umerica
lva l
u esaretoe ac
ho ther
.
d)Scie
ntificNo tati
on
Whe nr ecordinga nda n al
yzingn ume ri
cald atay ouma ye ncounte
rv eryl argen umbers(suc
ha s
987,000, 000, 000)o rv erys malln umb ers(sucha s0. 000000001 23).Si n
cet hesenumbersa r
e
di
fficul
tt owo rkwi tho renterint
oac ommo ncalcula
torweu sesci
e n
tifi
cn ot
a t
ion.Scie
nti
ficnot
ati
on
repre
sentsan umb erinthef o
llowi
ngfo rmat:
Nx1 0nwhe reN~1( N<1 0)a ndnist hen umb e
ro fspacesthedecimalpointismo v
e d
;totheleftifa
negat
ivenu mbera ndt other i
ghtifap osi
tivenumb e
r
1
1
Example:987 ,000,000, 000=9. 87x1 0a nd0. 00000000123=1 .23x1 0-9
e)Solu
tesa n
dSo lv
ents
Al i
qui
ds olut
ionisami xt
u r
eo ftwoormo resubstanceseve
nlydis
tri
b ut
edwi t
hineacho ther
.The
subs
tancewhicha c
tsastheliqu
idandcompri
sesthema jori
tyofthetotalvol
umei sc
a l
le
dt hesolv
ent
andtheo t
hersubstan
cedisso
lve
dinthesolve
ntisc a
lledthesolu
te.Typica
lly,theconcent
rati
onofa
sol
uti
oni n
dic
ateshowmu chsolu
teisdi
ssol
vedinsol
ventfo ragiv
envolume.
Forexamp l
e:Yo uhaveasolut
iont
hatis10mg/mL( NaCl/H2O)
·Thesodiumchlori
de(NaCl)i
sthesol
uteandthewa ter(H2O)isthesol
vent
·Theconcent
rati
ontell
susho wmuchsolut
e(10mg)i sdiss
olv
edineachmLo ft hesol
uti
on.
f)Concentra
tio
n s
Therearenumerouswaysofr ep
orti
ngconce
ntrati
on:
i
)Ma ss/volu
me( mg/mL).Ma sstovol
umes ol
uti
onsa recalc
ulat
edbyt heamo untofsolut
ed is
sol
ved
pervol
u meofsoluti
on.Fore xa
mple,i
fy ouha v
e10mg/mL( NaCl/H2O)s oluti
on,therearetenmg
ofsodi
umc hlor
idedis
sol
vedinonemLo fsol
ution
.
i
i)Pe r
c en
ts ol
uti
onsd e
termine
db ywe i
ght/volume.Pe rcen
t( %)sol
u t
ion
sb yweight/volu
mea re
3
TY
BMED31
8:BIOCHEMI
CALTECHNI
QUES
LECTURENOTES
I
n troduc
tion
Biochemist
r yi sthes t
u dyofc hemi calp rocessestha ttakep laceinl i
vingorga ni
smswi t
ht heult
ima te
aimo fu nder s
tan di
n gt hen atureo fl i
fei nmo l
eculart erms .Bi ochemicals t
u di
esa rea i
dedb y
avail
ab i
li
tyo fa n al
y t
icalt e
chn i
q uest ha ta dvancet hekn owledgeo ft hen a t
urea ndr e
lati
onships
betweenb i
o logi
ca lmo lecul
essu c
ha sn ucleicac i
dsa ndprotei
n sa ndtheircell
ularfu ncti
on.
I
d enti
ficat
io no fc onstitu
ents:e xp e
r imenta ldesi
gn
Exp eri
mentsa rec a refull
yd esign ed,e xecuteda ndd ataa nalyzedtoa nswe rs peci
ficq ues
tio
n so r
hyp o
theses.Ex perimen ta
ld e
signi nvolvesan umbe rofs t
age s:
·theidenti
fi cati
ono ft hes ubj
e c
tfo re xperime nt
ali nv
esti
ga ti
o n;
·thec ri
tic
a le v
alu a
tiono fthec urr ents ta
t eofkn owl e
dge( the‘ li
terature
’)oft hec hosensubje
cta rea
notingthes tr e
ngt hsa ndwe aknes seso ft heme t
ho dol
ogiesp reviousl
ya ppl
ieda ndthen ewhy pot
he ses
whiche mer gedfr omt hestudies
;
·thefo rmula ti
ono ft heq ues
tiono rhy pot
he sistob eaddressedb yt heplannedexperime n
t;
·thec are
fu ls e
lectiono ftheb iol
o gicals ystem( s
p eci
es,inv i
voo rinv i
tro)tob eusedfo rthestudy;
·theid e
ntificationo ft hevariablet ha tist ob estudie
d;t hec o n
siderat
ionoft heo therv a
riabl
est hat
willn ee
dt ob ec ontrolle
ds ot ha tt hes electedv aria
b l
ei st heo nlyfa ct
ort hatwi lld e
termin
et he
experimentalo utcome ;
·thed esi
gno ft hee xp er
imentin cludingt hes tatist
icalanalysiso fther esul
ts,c a
r efulevalu
ati
o no f
thema teri
a lsanda p paratustob eu seda ndt hec o
n sequ
entialpo tent
ialsafetyaspectso fthestudy;
·thee xecutiono ft hee xperi
me nti ncludinga pp
r opri
atec ali
b rati
onsa ndc o
ntrols
,wi thac arefully
wr i
ttenrecor doft heo ut
comes;
·ther epl
icationo ft hee xp
e r
imen ta sn ece
s saryfo rtheu namb iguousanalysi
so ftheo utcomes;
·t hee valua ti
ono ft heo utcome si nclud i
ngt hea p p
licati
o no fa ppr
op ri
ates tatis
tica
lt es
t st o
quantit
ativ
ed atawhe reappli
cab l
e ;
·thefo rmula ti
ono ft hema inconc lusi
o nstha tcanb edr a
wnfr o mther esu
lts;
·thefo rmula ti
ono fn ewhy pothes esa ndoffu turee xper
ime ntstha temergefr omt hes t
udy
Quant
it
a t
ivemeasu
rement
a)Uni
tsofme as
urement
TheFrenchSystè
meInter
nat
io
nalďUni
té
s(t
heSIs
yst
em)isthea
ccep
tedc
onve
nti
onforal
luni
tsof
meas
urement.Onemoleofasubs
tan
cei
sequ
alt
oit
smo l
ecul
armassex
pre
sse
dingrams,whe
rethe
1
,molecularma ssi sthes umo fthea tomicma sse
so ft hec onsti
t uen
ta t
oms.No tethatt het e
r m
molecularma ssisp referredtot heo ldertermmo lec
u l
arwe ight.TheSIu nitofc oncent
r at
ioni s
-3
expres
s e
di nt e
rmso fmo l
esp e
rc ubicme tre(molm )( Table1 )
.I np rac
ticethisisfa rtoolargefo r
3 -
3
normallabora t
oryp urpos
esa n
dau nitb ase
do nacu b
icd eci
me ter(dm,1 0 m)i spreferred.
Themo lari
tyo fas o
lutio
no fas u
b st
anc eexpres
sesthen u mb e
ro fmo l
esoft hesubstanceinonec u
b ic
deci
met e
ro fs ol
ution
.I ti
se xpr
essedb ythes ymbolM.
I
ts houldben otedthata t
omicandmo lecularma s
sesa rebo thexp re
s s
edinDa lton
s( Da)orkiloDa lt
on s
(kDa),whe reo neDa l
tonisa natomicma ssu ni
tequalt oon etwe l
ft hoft hema sso fonea t
omo ft he
1
2
Ci sotope
.Ho we v
er,b io
chemist
sp refert ouset het er mr elativ
emo le
cularma ss( Mr).Thi si s
1
2
defin
eda st hemo lecul
arma ssofas ubstancerelat
ivet oo ne-t
we lftho fthea tomicma ssoft he C
i
s o
tope.Mrt hereforehasn ouni
ts.
Thust herelativ
emo lecula
rma sso fsodiumc hlo
rideis23( Na)p lus35. 5(Cl)i.e.58. 5,sot haton e
moleis58. 5gr ams.I fthiswasdissolvedinwa t
eran da d
ju s
t e
dt oat ota
lv ol
umeo f1d m3t hesolut
io n
wouldb eo n emo l
ar( 1M) .Bi o
logicals ubsta
ncesa r emo s
tfr equen
tlyfo unda tr el
ati
velyl ow
conce
ntrati
on sa ndini nv itr
omo de
ls y s
temst hev o
lume so fs tocks olut
ionsr egularl
yu sedfo r
experi
me nt
alp urposesarealsosmall.Thec onseque
ncei stha texperimentalsolu
tionsareu s
uallyinthe
mM,n Ma ndp Mr anger a
therthanmo lar.
Table1:SIu ni
ts-Basicuni
ts
Qu
ant
it
y SIu
nit Sy
mbo
l
Le
ngt
h Me
tre m
Ma
ss Ki
lo
gra
m kg
Ti
me s
eco
nds s
El
ect
ricc
urr
ent Amp
ere A
Te
mpe
rat
ure Ke
lvi
n K
Lu
min
ousi
nte
nsi
ty Ca
nde
la c
d
Amo
unto
fsu
bst
anc
e Mo
le mo
l
I
nscie
nceitiscri
ti
caltha
texper
iment
sandtheob
ser
vati
onsrec
orde
dar
erepea
tabl
e.
There
forequ a
nti
tat
iveobser
vati
onsaremu c
hmo r
ev a
luabl
et ha
nqual
it
ati
ve.Whenan ume
ric
al
val
ueisgi v
ent oac hara
cte
ris
ticital
lowsfordire
ctc o
mparis
onbe
tweenobs
erv
ati
onsmad
eb y
2
, di
fferentp eopleora tdifferentti
mes .
Qu a
nti
tativeme asuremen tiscoll
ecti
ngi nformationthatisnumericinn a
ture.
Qu a
nti
tatived ataha sv al
ueso fav ariabledifferi
ngi na mountinn ume r
ict ermsr a
therthanin
desc
ript
ivet erms.
Qu a
nti
tativeme t
hodsa rer es
earchte chniquesthatareu se
dt ogatherq ua
n t
itat
ived a
ta-in
formati
on
deal
ingwi thn umbersa nda nyt
hingtha tisme asurable
.St at
isti
cs,tablesandgr aphs,areofte
nu s
ed
topresentt her esul
t soft heseme thod s
.The ya r
et here
foret ob ed i
st
inguishedfromq ual
it
ativ
e
methods
.
c)Accuracy&Pr ecision
Accurac
ya n dp reci
sions houldb ec o nsi
deredwhe therc oll
ect
ingd at
ao rme asuri
ng mater
ials
.
Accuracyr eferstoho wc lo
seame asure dvalueistothet r
uev al
ue.
Preci
sio
nr eferstoho wr eproduc
ibleame a
surementis.Ify our epeatame asurementan umberof
ti
mes,itisho wc losethen umerica
lva l
u esaretoe ac
ho ther
.
d)Scie
ntificNo tati
on
Whe nr ecordinga nda n al
yzingn ume ri
cald atay ouma ye ncounte
rv eryl argen umbers(suc
ha s
987,000, 000, 000)o rv erys malln umb ers(sucha s0. 000000001 23).Si n
cet hesenumbersa r
e
di
fficul
tt owo rkwi tho renterint
oac ommo ncalcula
torweu sesci
e n
tifi
cn ot
a t
ion.Scie
nti
ficnot
ati
on
repre
sentsan umb erinthef o
llowi
ngfo rmat:
Nx1 0nwhe reN~1( N<1 0)a ndnist hen umb e
ro fspacesthedecimalpointismo v
e d
;totheleftifa
negat
ivenu mbera ndt other i
ghtifap osi
tivenumb e
r
1
1
Example:987 ,000,000, 000=9. 87x1 0a nd0. 00000000123=1 .23x1 0-9
e)Solu
tesa n
dSo lv
ents
Al i
qui
ds olut
ionisami xt
u r
eo ftwoormo resubstanceseve
nlydis
tri
b ut
edwi t
hineacho ther
.The
subs
tancewhicha c
tsastheliqu
idandcompri
sesthema jori
tyofthetotalvol
umei sc
a l
le
dt hesolv
ent
andtheo t
hersubstan
cedisso
lve
dinthesolve
ntisc a
lledthesolu
te.Typica
lly,theconcent
rati
onofa
sol
uti
oni n
dic
ateshowmu chsolu
teisdi
ssol
vedinsol
ventfo ragiv
envolume.
Forexamp l
e:Yo uhaveasolut
iont
hatis10mg/mL( NaCl/H2O)
·Thesodiumchlori
de(NaCl)i
sthesol
uteandthewa ter(H2O)isthesol
vent
·Theconcent
rati
ontell
susho wmuchsolut
e(10mg)i sdiss
olv
edineachmLo ft hesol
uti
on.
f)Concentra
tio
n s
Therearenumerouswaysofr ep
orti
ngconce
ntrati
on:
i
)Ma ss/volu
me( mg/mL).Ma sstovol
umes ol
uti
onsa recalc
ulat
edbyt heamo untofsolut
ed is
sol
ved
pervol
u meofsoluti
on.Fore xa
mple,i
fy ouha v
e10mg/mL( NaCl/H2O)s oluti
on,therearetenmg
ofsodi
umc hlor
idedis
sol
vedinonemLo fsol
ution
.
i
i)Pe r
c en
ts ol
uti
onsd e
termine
db ywe i
ght/volume.Pe rcen
t( %)sol
u t
ion
sb yweight/volu
mea re
3
