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Prote in De terminat ion by UV Absorpt ion

A l a s t a i r A i t k e n a n d M i c h e l e L e a r m o n t h

1 I n t r o d u c t i o n

1 .1 . N e a r U V A b s o r b a n c e 2 8 0 n m )

Qua nt i ta t i on o f t he am oun t o f p ro t e in i n a so lu t i on is poss ib l e i n a s imple spe c t rom-

e t er. Ab sorp t ion o f r ad i a t i on i n t he near UV by p ro t e ins dep ends on the Tyr and Trp

con ten t ( and to a very smal l ex t en t on t he amo un t o f Phe and d i su l f ide bonds) . T here-

fore the 4 2 8 0 var i es g rea t l y be tween d i f f e ren t p ro te ins ( fo r a 1 m g/m L so lu ti on , f rom 0

u p t o 4 f o r s o me t y r o s i n e - r i ch w o o l p r o t e i n s , a l t h o u g h m o s t v a l u es a r e i n t h e r an g e

0 . 5 - 1 . 5 [ 1 ] ) . The advan tages o f th i s method are tha t i t i s s imple , and the samp le i s

r ecoverab l e . The method has some d i sadvan tages , i nc lud ing in t e r f e rence f rom o ther

ch romophores , and the spec i f i c abso rp t ion va lue fo r a g iven p ro t e in mus t be de t e r -

mined . The e x t inc t ion o f nuc l e i c ac id in the 280-n m reg ion m ay be as mu ch as 10 t imes

tha t o f p ro t e in a t t he i r same w aveleng th , and hence , a f ew percen t o f nuc l e i c ac id can

great ly inf luence the absorpt ion.

1 .2 . F a r U V A b s o r b a n c e

The pep t ide bo nd absorbs s t rong ly i n t he f a r UV wi th a m ax im um a t abou t 190 nm.

Th i s very s t rong absorp t ion o f p ro t e ins a t these w aveleng ths has been u sed in p ro t e in

de t ermina t ion . B ecause o f the d i f f icu l t ies caused by absorp t ion by ox yge n and the l ow

outpu t o f conven t iona l spec t ropho tometer s a t t h is wavel eng th , mea surem en t s a re mo re

conve n ien t ly mad e a t 205 nm, whe re t he absorbance i s abou t ha l f t ha t a t 190 nm. M os t

p ro t e ins have ex t inc t i on coef f ic i en t s at 205 nm fo r a 1 m g/m L so lu t ion o f 30 -3 5 and

b e t w een 2 0 an d 2 4 a t 2 1 0 n m

2 ) .

Va r ious s ide cha ins , i nc lud ing those o f Trp , Phe , Tyr , His , Cys , M et , and A rg ( i n

that descending order) , make cont r ibut ions to the A205 3 ) .

The advan tages o f t h is m ethod inc lude s impl i c i t y and sens i t iv i t y . As i n the m ethod

out l ined in S ect ion 3 .1 . the sam ple i s recov erable and in addi t ion there i s l i tt l e var iat ion

in response be tw een d i f f e ren t p ro t e ins , perm i t ti ng near -abso lu t e de t e rm ina t ion o f p ro -

t e in . D i sadvan tag es o f th i s metho d inc lude t he necess i t y fo r accura te ca l i b ra t ion o f t he

spec t roph o tom eter i n t he f a r UV. M any buf fer s and o ther com ponen t s , such as hem e o r

pyr idoxal groups, absorb s t rongly in th i s region.

From:

The Prote in Protocols Handboo k

Edi ted by: J . M. Walker H uma na Press Inc. Totowa NJ

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  Aitken and Learmonth

2 M a t e r i a l s

1 . 0 . 1 M K 2 S O

4

(pH 7.0).

2 . 5 m M p o t a s s i u m p h o sp h a t e b u f fe r , p H 7 .0 .

3 . No nionic de t e rgen t (0 .01 Br i j 35)

4 . Guanid in ium-HC1.

5 . 0 .2-1am Mi l l i pore (W at ford , UK ) f i lt e r.

6 . U V -v i s ib l e sp ec t ro m e t e r : T h e h y d ro g en l am p sh o u l d b e s e lec t ed fo r m a x i m u m i n t en si t y

a t t he par t i cu l a r wav elength .

7 . Cuvet s , quar t z , fo r <215 nm.

3 M e t h o d s

3 .1 . Es t im a t io n o f Pro t e in b y N e a r U V A b s o r b a n c e 2 8 0 n m )

1. A re l i ab l e spec t rop hotom eter i s necessary . The pro t e in so lu t ion mu st be d i lu t ed in the

buffe r to a conce nt ra t ion tha t is wel l wi th in the accura t e range o f t he ins t rume nt

s ee

No tes 1 and 2) .

2 . The pro t e in so lu t ion to be mea sured c an be in a wide range of buffe rs , so it i s usua l ly no

pro blem to f ind one tha t i s approp r i a t e for t he pro t e in which m ay a l read y be in a par t i cu l a r

buffe r requi red for a pur i f ica t ion s t ep or assay for enzy me ac t iv i ty , fo r exam ple

see

N o t es

3 and 4) .

3 . M easur e the absorb ance o f t he pro t e in so lu t ion a t 280 nm, us ing quar t z cuve t s o r cuve t s

tha t a re know n to be transpa ren t t o th i s waveleng th , f i ll ed wi th a vo lum e o f so lu tion suf f i -

c i en t t o cover t he aper ture t h roug h wh ich the l igh t beam p asses .

4 . The v a lue ob ta ined w i l l depe nd on the pa th l ength of t he cuve t . I f no t 1 cm, i t mu st be

adjus t ed b y the appropr i a t e fac tor . The Bee r-Lam ber t l aw s t a t es tha t :

A (abso rbanc e) = e c / (1)

wh ere ~ = ex t inc t ion co eff i c i en t, c = conce nt ra t ion in mol /L an d l = op t ica l pa th l en gth in

cm . T h e re fo re , i f e i s k n o w n , m easu rem en t o f A g i v e s t h e co n cen t r a t i o n d i r ec tl y , e i s

norm al ly quoted for a 1 -cm pa th l ength .

5 . T h e ac t u a l v a l u e o fU V ab so rb an ce fo r a g i v en p ro t e in m u s t b e d e t e rm i n ed b y so m e ab so -

l u te m e t h o d , e . g ., c a l cu la t ed f ro m t he am i n o ac i d co m p o s i t io n , w h i ch can b ed e t e r m i n ed

b y am i n o ac i d an a l y s i s

4) .

T h e U V ab so rb an ce fo r a p ro te i n i s th en ca l cu l a t ed acco rd i n g

to the fo l low ing formu la :

A280 (1 mg /mL ) = (5690nw + 1280n y +

120nc) / M

(2)

wh ere n w, ny, an d n c a r e t h e n u m b er s o f T rp , T y r , an d C y s r e s id u es i n t h e p o l y p ep t i d e o f

ma ss M and 569 0, 1280 and 120 are the respec t ive ex t inc t ion coeff i c i en t s for t hese res i-

d u es

s ee

No te 5) .

3 . 2 . Es tim a t io n o f Pr o te in b y F a r U V A b s o r b a n c e

1. Th e pro t e in so lu t ion i s d i lu t ed wi th a sodium chlor ide so lu t ion (0 .9 w/v) un t i l t he

ext inc t ion a t 215 nm i s <1 .5

see

Notes 1 and 6) .

2 . Al t e rna t ive ly , d i lu t e t he sam ple in another non -UV -abso rb ing buffer such as 0 .1MK 2SO4,

co n t a i n in g 5 m M p o t a s s i u m p h o sp h a t e b u f f e r ad j u s ted t o p H 7 . 0

s ee

Note 6) .

3. M easu re the absorb ances at the appropriate wav elengths (ei ther A280an d A205,or A225 and A215,

depe nding on the fo rmu la to be appl ied), using a spect rom eter fi t ted wi th a hydroge n lamp that

i s a ccu ra t e a t t h e se w av e l en g t h s , u s i n g q u a r t z cu v e t s f i l l ed w i t h a v o l u m e o f so l u t i o n

sufficient to cover the aperture through w hich the l ight be am pa sses (de tai ls in Sect ion 3.1. ) .

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UV Absorption 5

4. The

A2o 5

for a 1 mg /mL so lu t ion of p ro t e in

(,42051

mg/mL)can be ca l cu la t ed w i th in +2 ,

a cco rd i n g t o t h e em p i r i c a l f o rm u l a p ro p o sed b y S co p es

2 ) s e e

N o t es 7 -1 0 ) :

A2051 mg/mL = 27 + 120 (A280/A205) (3)

5 . A l t e rn a ti v e l y , m easu rem en t s m ay b e m ad e a t l o n g e r w av e l en g t h s

5 ) :

Protein concent rat ion (~tg/mL) = 144

(,4215

A 2 2 5 )

(4)

The ex t inc t ion a t 225 nm i s sub t rac t ed f rom tha t a t 215 nm; the d i f fe rence mul t ip l i ed by

144 gives the protein concen t rat ion in the sample in ~g/m L. W ith a part icular protein und er

spec i f i c condi t i ons accura t e m easurem ent s o f conc ent ra t ion to wi th in 5 ~ tg /L are poss ib l e .

4 N o t e s

l . I t i s bes t t o me asure absorban ces in t he range 0 .05-1 .0 (b e tween 10 and 90 of t he inc i -

dent rad i a t ion) . At a roun d 0 .3 absorban ce (50 absorp t ion) , t he accu racy is g rea t es t .

2. Bov ine serum album in i s frequent ly used as a protein standard; 1 mg /mL has an A280 of 0.66.

3. If the solut ion i s turbid, the appare nt A280 wi l l be increas ed by l ight scat tering. F i l t rat ion

( through a 0 .2-~tm Mi l l i pore f i lt e r ) o r c la r i f i ca tion o f t he so lu t ion by cen t r i fuga t ion can be

car r i ed ou t . For t u rb id so lu t ions , a convenien t approximate cor rec t ion can be appl i ed by

subt rac t ing the A310 (pro t e ins do not no rmal ly a bsorb a t th i s wav eleng th un less t he y co n-

t a in par t i cu l a r c hrom opho res) f rom the A280.

4 . At l ow concent ra t ions , p ro t e in can be los t f rom so lu t ion by adsorp t ion on the cuve t ; t he

h igh ion ic s trength he lps to preven t t h is . Inc lus ion ofa nonionic de t e rgent (0 .01 Br i j 35)

in the bu ffer m ay a l so he lp to preven t t hese losses.

5 . The presen ce o f nonp ro te in chrom opho res (e .g ., heme, pyr idoxal ) can increase A28o. I f

nuc le i c ac ids a re presen t (wh ich absorb s t rongly a t 260 nm ) , the fo l lowing form ula can be

appl i ed . Thi s g ives an accu ra t e es t imate of the pro t e in conten t by rem oving the cont r ibu-

t ion to absorban ce by nuc leo t ides a t 280 nm , by measu r ing the

A260

wh ich i s l a rge ly owing

to the lat ter

6 ) .

Prote in (mg/mL) = 1 .55

A280- 0 . 7 6

A260 (5)

Other formulae (us ing s imi l a r p r inc ip l es of absorbance d i f fe rences) employed to de t e r -

mine p ro t e in in the poss ib l e presence of nuc le ic ac ids a re the fo l lowing

7 , 8 ) :

Prote in (mg/mL)

= (A235 -

A280)/2.51

(6)

Pro te in (mg/m L) = 0 .183

A230 -

0.075.8

A260 (7)

6 . Pro t e in so lu t ions obe y Beer-L am ber t ' s La w a t 215 nm provided the ex t inc t ion i s <2 .0 .

7 . S t r i c tl y speaking , t h i s va lue appl ies t o t he pro t e in in 6M guanid in ium-H C1, bu t the va lue

in buffe r i s gene ra l ly wi th in 10 of t h is va lue , and the re l a t ive absorba nces in guani -

dinium-H C1 and buffe r can be easi ly determined by paral lel di lut ions from a stock solut ion.

8 . Sod ium chlor ide , am mo nium su l fa t e , bora t e , phosph ate , and Tr i s do not i n t e r fe re , wh ereas

0 .1M aceta t e , succ ina t e , c i tra t e , ph tha l a t e , and barb i tura t e show h igh abso rp t ion a t 215 nm.

9 . T h e ab so rp t i o n o f p ro t e in s i n th e r an g e 2 1 5 -2 2 5 n m is p r ac ti c a l ly i n d ep en d en t o f p H

b e t w een p H v a l u e s 4 -8 .

10 . The spec i f i c ex t inc tion coeff i c i en t o f a num ber ofpro t e ins and pept ides a t 205 nm and 210

n m

3 )

has been de t e rmined . The average ex t inc t ion coeff i c i en t for a 1 m g/m L so lu t ion o f

40 se rum pro te ins a t 210 nm i s 20 .5 + 0 .14 . At th i s waveleng th , a p ro t e in con cent ra t ion of

2 ~tg/mL gives A = 0.04

5 ) .

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6 Aitken and Learmonth

R e f e r e n c e s

1. Kirsche nbaum , D. M. (1975) M olar absorptivity and Al /1 cm values for proteins at

selected wa velengths of the ultraviolet and visible regions.

A n a l B i o c h e m .

68, 465--484.

2. Scopes, R. K. (1974) Measu reme nt of protein by spectrometry at 205 nm. A n a l . B i o c h e m .

59 , 277-282 .

3. Goldfarb, A . R., Saidel , L. J . , and Mosovich, E. (1951) The ultraviolet absorption spectra

of proteins. Jr. Biol. Chem. 193, 397-404.

4. Gil l , S . C. and von Hippel, P . H. ' (1989) Calculation o f protein extinction coefficients from

amino acid sequence data. A n a l . B i o c h e m . 182, 319--326.

5. W addell , W. J. (1956) A simple UV spe ctrophotometric m ethod for the determination o f

protein . Jr. Lab. Clin . M ed . 48, 311-314.

6. Layne, E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins.

M e t h o ds E n z y m o l. 3 , 447-454 .

7. W hitaker, J . R. and Granum, P. E. (1980) An absolute method for protein determination

based on difference in absorbance at 235 and 280 nm.

A n a l . B i o c h e m .

109, 156-159.

8. Kalb, V. F. and Bernlohr, R. W. (1977). A new spectrophotometric a ssay for protein in cell

extracts. A n a l . B i o c h e m . 82 , 362-371 .