LAS CLÁUSULAS RESTRICTIVAS DE RESPONSABILIDAD DEL PROVEEDOR

HSA = Human Serum Album in

BSA = B ovine Serum Album in

SDS = Sodium Dodecyl S u lph a te

CPMAS = Cross P o la r is a tio n Magic Angle S p in n in g

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The unoonjugated b il i r u b i n IXoc -z , z formed from the breakdown o f haem oglobin^^^ shows e x tre m e ly low s o lu b il it y in w ater; i t i s

“ 9 —3 (2 )

c a lc u la te d to be in th e o rd e r o f 10“ mol dm” . T h is e x tre m e ly low s o lu b il it y is a ttr ib u te d to th e e x te n s iv e in tra m o le c u la r hydrogen

bonding, o f b i lir u b in (see Figure 1 ).

CH

CH _CH

F ig u re 1 In tra m o le c u la r hydrogen bonding o f B ilir u b in .

A t pH 7 .2 th e hydrogen bonding i s so e x te n s iv e th a t a l l the bonding m o ie tie s o f th e m o le cu le a re s a tu ra te d , le a v in g th e h y d ro - p ho b ic groups a t th e outer aspect o f th e m olecule. As a re s u lt o f i t s aqueous in s o lu b il it y , near p h y s io lo g ic a l pH, non co n ju g a te d b ilir u b in

a c id ten d s to form a g g r e g a t e s . A t pH 7 .4 -8 .0 B rodersen^^^ has observed c o llo id b e h a vio u r o f b ilir u b in a c id . The n e u ro to x ic ity o f b ili r u b i n ( k e r n ite r u s ) can th e re fo re be a d e q u a te ly e x p la in e d by t h is

behaviour. The in tra m o le c u la r hydrogen bonding re n d e rs the m o le cule in s o lu b le i n the plasm a. Ihe lip o p h i l i c m o ie tie s become exposed and

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b ra iie s o f th e c e lls d is r u p tin g th e membranes s tr u c tu r e and ham pering th e a c t i v it y o f th e a tta c h e d enzymes» In norm al c o n d itio n s t h is lip o p h il ic behaviour le a d in g to n e u r o to x ic ity i s never observed sin ce a l l the b i l i r u b i n d ia n io n s c ir c u la t e i n th e b lo od t i g h t l y bound to serum album in»

In s p ite o f the f a c t th a t serum album in has been one of th e most th o ro u g h ly s tu d ie d p ro te in s , i t s th re e d im e n sio n a l s tr u c tu r e has n o t y e t been determ ined. T h is p r o te in c o n ta in s only one peptide ch a in

Helices Subdomains 1 A -B Domains 1 C 2 A-B 2 C 3 A -B 3 C One ^ Two " Three

F ig u re .

2

th e model o f Brown. ( 5 , 6 )

w ith some 580 amino a c id residues (see Appendix 2 fo r f u l l lis t in g )

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( 7 )

The c h a in i s fo ld e d in a unique p a tte rn o f double lo o p s .

Recent m odels o f alb um in m o le cu le s (F ig u re 2) i l l u s t r a t e th e p ro te in s secondary s tr u c tu r e as h a v in g th re e s o -c a lle d dom ains; a n io n b in d in g s it e s and s ix subdom ains. Each subdomain c o n ta in s th re e p a r a lle l CC h e lic e s , x ,y , and z , each h a vin g a p p ro x im a te ly 22 amino a c id re s id u e s . T h is model can e x p la in many o f th e b in d in g ch a ra c­

t e r i s t i c s . As d iscu sse d p re v io u s ly (C h a pte r 4, p. 104) a lb u m in i s a u n iv e rs a l p r o te in c a r r ie r , b in d in g in o rg a n ic io n s , o rg a n ic a n io n s, and uncharged s p e c ie s . In 1929 B ennhold^^^ was th e f i r s t to emphasize the b io lo g ic a l im p o rta nce o f u nco n ju g a te d b in d in g to a l b u m i n i n

u n d e rs ta n d in g b i l i r u b i n tra n s p o rt and t o x i c i t y .

The secondary b in d in g s it e o f th e b i l i r u b i n diand.on i s between th e h a lf domains 3A-B.

J a c o b s e n ^ f o u n d th a t b i l i r u b i n i s bound to a lb u m in a t a s in g le h ig h a f f i n i t y s it e and o b ta in e d e vidence o f two weaker s ite s w ith

f 11 )

a f f i n i t i e s 10 to 100 f o ld lo w e r. B ro d e rs e n 's s to ic h io m e tr ic s tu d ie s have fou n d th a t th re e b i l i r u b i n d ia n io n s a re in te r n a lis e d w it h in a m o le cu le o f human serum alb um in (HSA). Techniques o f a f f i n i t y l a b e l l i n g ^ c h e m i c a l m o d ific a tio n , and cleavage o f p r o te in s by enzym atic h y d r o l y s i s ^ a l l in d ic a te th a t f o r b oth bovine and huraan serum alb um in re g io n 2 A-B and a sm a ll p o r tio n o f subdomain 10 (see F ig u re 2) i s th e h ig h a f f i n i t y b in d in g s it e f o r