SECTOR NORTE PAYLLA
4.2.2. Análisis de la estructura del rito andino
3.2.3.1 G en eral P ro p er ties o f th e D 8 4 N M u tan t
E. coli cells expressing the m utant D84N porphobilinogen deam inase
were grow n in the same m anner as the cells expressing the m utant D84E enzyme. The m utant D84N enzyme was then also purified using the sam e protocol as that used for the m utant D84E enzyme.
As w ith the D84A m utant, the solid pure D84N m utant was found to be slightly pink. Approxim ately 25mg of pure protein was obtained per prepararation from 2L of media. The purity of the enzyme w as studied at various stages of the purification procedure by polyacrylam ide gel
electrophoresis using a 12% SDS gel. The molecular mass of the protein, 35,000 Da, was determ ined by comparing the mobility on the gel w ith th at of the molecular mass markers.
3 .2.3.2 F.P.L.C. o f th e D 8 4 N M u tan t
After purification the m utant D84N porphobilinogen deam inase (lO m g/m l) w as applied to a MonoQ H R 5/5 column attached to a
Pharm acia f.p.l.c. system. The M onoQ colum n had been equilibriated in 20mM T ris/H C l buffer pH 7.5 containing 7mM p-m ercaptoethanol. The purified protein w as eluted from the colum n in the sam e buffer u sing a linear salt gradient. The resulting chrom atogram is presented in figure 3.10.
The chrom atogram in figure 3.10 shows that the f.p.l.c. system allow ed the further separation of protein by anion-exchange
chrom atography. M ultiple peaks were observed on the chrom atogram , eluting at Im M NaCl, 165mM NaCl, 185mM NaCl, 205mM NaCl, 225mM NaCl w ith a shoulder at 235mM NaCl, 270mM NaCl and at 290mM NaCl.
Figure 3.10 F.P.L.C. Trace of Purified D84N M utant Porphobilinogen D eam inase Absorbance of Protein Salt Gradient (D Ü 8 < 165m M 185m M 2 0 5 m M 2 2 5 m M 235m M NaCl NaC l NaCl NaCl NaCl
27 0 m M 2 9 0 m M NaCl NaCl Im M NaCl - 300 - 200 100 0 40
ih
O D Fractions3.2.3.3 P o ly a c ry la m id e G el E lectro p h o resis o f th e P ea k s Iso la te d F rom th e F.P.L.C. o f D 8 4 N
The fractions isolated from the f.p.l.c. of D84E were analysed by denaturing and non denaturing polyacrylamide gel electrophoresis, to examine further their deam inase content. The gels are show n in figures 3.11 and 3.12.
The denaturing gel in figure 3.11 shows that lanes 1-9 contained little deaminase, lanes 10-15 contained a strong band that m igrated the same distance as the 36,000 Da band of the molecular w eight m arker and lanes 16- 22 contained a band that m igrated the same distance as the 36,000 Da band of
C h a p te r 3 : C h a racterisatio n of the A sp-84 M u ta n ts
F ig u re 3.11 T h e D e n a tu rin g P o ly a c ry la m id e G els of th e F ra c tio n s C o llected fro m th e F.P.L.C. of th e D 84N M u ta n t
T h e la n e c o n ta in in g th e h o lo e n z y m e (w ild -ty p e co n tro l) a n d th e m o le c u la r m a ss m a rk e r are la b e lle d 'W T' a n d 'M k' resp ectiv ely .
A ll o th e r la n es c o n ta in c o n se c u tiv e fractio n s iso la te d fro m th e f.p.l.c. c o lu m n , a n d a re n u m b e re d a c c o rd in g to th e c h ro m a to g ra m s h o w n in fig u re 3.10. KDa 66 45 36 29 24 20.1 14.2 WTMk 1 2 3 4 5 6 7 8 9 10 11 KDa WTMk 12 13 14 15 16 17 18 19 20 21 22 66 45 36 g 20.1 14.2
1
th e m o le c u la r m a ss m a rk e r, as w ell as se v e ra l o th e r p ro te in s . T h is in d ic a te d th a t th e p e a k s o b s e rv e d o n th e c h ro m a to g ra m e lu tin g at 205, 225, 270 a n d290m M N aC l p ro b a b ly all c o n ta in e d d e a m in a s e , a lth o u g h th e la st tw o p e a k s m a y n o t h a v e re s u lte d fro m d e a m in a s e alone.
T h e fractio n s b e lie v e d to c o n ta in d e a m in a s e w e r e th e n su b je c te d to e le c tro p h o re s is o n a n o n - d e n a tu r in g gel, th e re s u lts of w h ic h are s h o w n in fig u re 3.12.
F ig u re 3.12 T he N o n -D e n a tu rin g P o ly a c ry la m id e G el o f th e P e a k s O b se rv e d from th e F.P.L.C. of th e D 84N M u ta n t
T h e la n e c o n ta in in g th e a p o e n z y m e a n d h o lo e n z y m e (w ild -ty p e c o n tro ls) are lab elle d 'A p o ' a n d 'W T' acc o rd in g ly . F or clarity , th e m ig ra tio n s of th e
a p o e n z y m e a n d h o lo e n z y m e a re m a rk e d in th e fig u re.
A ll o th e r lan es c o n ta in c o n se c u tiv e fractio n s is o la te d fro m th e f.p.l.c. c o lu m n , a n d are n u m b e re d a c c o rd in g to th e c h ro m a to g ra m s h o w n in fig u re 3.10.
A d o W T 10 11 12 13 14 15 16 17 18 19 20
- a p o e n z y m e
C -<--- h o lo e n z y m e (E)
T h e n o n d e n a tu r in g gel in fig u re 3.12 s h o w s th a t la n e 10 c o n ta in e d a p ro te in w ith th e s a m e e le c tro p h o re tic p ro p e rtie s as w ild -ty p e . L an e 11
c o n ta in e d p r o te in w ith a n u m b e r of b a n d s th a t m ig r a te d to th e s a m e d is ta n c e a n d b e y o n d th e w ild -ty p e . L an es 12-17 also c o n ta in e d p r o te in w ith b a n d s th a t m ig ra te d b e y o n d w ild -ty p e . L an es 19 a n d 20 a p p e a r e d to c o n ta in a p ro te in b a n d th a t m ig ra te d slig h tly fu rth e r th a n th o se s e e n in la n e 12-17.