Fase 5: Evaluar la calidad interna final

5.2.2.2.1 V isu a lis a tio n o f th e R e co n stitu tio n o f H o lo e n z y m e fro m A p o e n z y m e a n d th e E ffect o f P ro teo ly tic D ig e s tio n , b y

P o ly a c r y la m id e G el E lectro p h o resis

The apparent instability of apoenzyme to heat treatm ent suggests that it will also have an increased susceptibility to proteolytic digestion. Trypsin, a proteolytic enzym e which cleaves on the carboxyl side of arginine and lysine residues, is likely to have access to more residues in an unfolded structure than in a folded one. The susceptibility to trypsin digestion of apoenzyme, holoenzyme and holoenzyme reconstituted from apoenzyme was therefore m onitored. Trypsin has previously been found to cleave the holoenzyme rapidly at lysine 64 in the sequence FVKELE.

A poenzym e alone and apoenzyme which had been preincubated w ith a fifty m olar excess of porphobilinogen at 37°C for one hour were subjected to trypsin digestion (Im g /m l) at 37°C for thirty minutes. Both samples, together w ith their undigested counterparts, were analysed by both denaturing and non denaturing gel electrophoresis. For comparison, holoenzyme w as also subjected to the sam e conditions and analysed. The results are show n in figures 5.16 and 5.17.

The gel in figure 5.16 shows that the apoenzyme (lane 7), which in this case w as not entirely pure, was seen as a band which m igrated the same distance as the 36kDa standard on the molecular mass marker. On trypsin digestion (lane 8), the band was completely lost and replaced by two major bands, m igrating to the same distance as the 24kDa and 12kDa standards on the m olecular mass m arker (representing a cleavage betw een arg-131 and arg- 132). Further bands m ay have been present at molecular weights below 12kDa. Apoenzym e which had been substrate-preincubated and then subjected to trypsin digestion (lane 10), was seen to m aintain the 36 KD band, w ith

additional bands observed at 24kDa and 12kDa, and m ultiple bands betw een 30-36kDa and below 12kDa.

H oloenzym e which had not been subjected to digestion ran as a single band w hich m igrated to the same distance as the 36kDa standard on the

F ig u re 5.16 D e n a tu rin g P o ly a c ry la m id e G el S h o w in g th e S u sc e p tib ility of H o lo e n z y m e a n d A p o e n z y m e to T ry p s in D ig estio n

T h e la n e s c o n ta in in g th e m a rk e r are in d ic a te d w ith 'M k ' L an e 1 c o n ta in s th e w ild -ty p e h o lo e n z y m e

L an e 2 c o n ta in s try p s in -d ig e s te d w ild -ty p e h o lo e n z y m e

L an e 3 c o n ta in s w ild -ty p e h o lo e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n L an e 4 c o n ta in s w ild -ty p e h o lo e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n a n d th e n try p s in -d ig e s te d

L an e 5 c o n ta in s w ild -ty p e a p o e n z y m e

L an e 6 c o n ta in s try p s in -d ig e s te d w ild -ty p e a p o e n z y m e

L an e 7 c o n ta in s w ild -ty p e a p o e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n L an e 8 c o n ta in s w ild -ty p e a p o e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n a n d th e n try p s in -d ig e s te d

KDa

14.2

20.1

m o le c u la r w e ig h t m a rk e r for b o th th e free e n z y m e (lan e 2) a n d s u b s tra te - p r e in c u b a te d e n z y m e (lane 4). B oth s a m p le s b e h a v e d sim ila rly u p o n try p s in d ig e s tio n (lan es 3 a n d 5), d is p la y in g th e s a m e b a n d in g p a tte r n as for d ig e ste d s u b s tr a te - p r e in c u b a te d a p o e n z y m e (lan e 1 0).

T he free a p o e n z y m e is th e re fo re u n a b le to m a in ta in th e te rtia ry s tr u c tu re u p o n tr y p s in d ig e stio n , in c o n tra s t to h o lo e n z y m e . T he

Chapter 5 ; Characterisation of Apoenzyme

a p o e n z y m e h a s clearly b e e n d ig e s te d to n u m e ro u s lo w m o le c u la r m a ss fra g m e n ts. H o w e v e r, re c o n s titu te d a p o e n z y m e is seen to d is p la y a n id e n tic a l p a tte r n of b a n d s to th a t of h o lo e n z y m e a n d th u s m u s t b e a d o p tin g a m o re tig h tly fo ld e d n a tiv e c o n fo rm a tio n .

F ig u re 5.17 N o n D e n a tu rin g P o ly a c ry la m id e G el S h o w in g th e S u sc e p tib ility of A p o e n z y m e to T ry p s in D ig estio n

L an e 1 c o n ta in s th e w ild -ty p e h o lo e n z y m e .

L an e 2 c o n ta in s try p s in -d ig e s te d w ild -ty p e h o lo e n z y m e .

L ane 3 c o n ta in s w ild -ty p e h o lo e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n . L ane 4 c o n ta in s w ild -ty p e h o lo e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n a n d th e n try p s in -d ig e s te d .

L an e 5 c o n ta in s w ild -ty p e a p o e n z y m e .

L an e 6 c o n ta in s try p s in -d ig e s te d w ild -ty p e a p o e n z y m e .

L an e 7 c o n ta in s w ild -ty p e a p o e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n . L ane 8 c o n ta in s w ild -ty p e a p o e n z y m e , in c u b a te d w ith p o rp h o b ilin o g e n a n d th e n try p s in -d ig e s te d . 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 ure. 1 2 3 4 5 6 7 8 -a p o e n z y m e ^ ^ --- h o lo e n z y m e (E) * 4

The gel in figure 5.17 shows that the holoenzyme, upon trypsin digestion (lane 2), ran as a double band m igrating further than that of

untreated holoenzyme. Substrate-preincubated holoenzym e (lane 3)migrated as m ultiple bands m igrating to the sam e distance and beyond that of untreated holoenzyme (lane 1), although on trypsin digestion (lane 4) only the bands m igrating beyond untreated holoenzyme were observed. The characteristic ladder of bands of apoenzyme (lane 5) were lost on trypsin digestion for apoenzym e (lane 6). Substrate-preincubated apoenzym e (lane 7) gave rise to a typical profile of a ladder w ith additional bands m igrating further than those of free holoenzyme. On trypsin digestion, the ladder of substrate-preincubated apoenzym e was lost (lane 8) giving rise to a banding pattern similar to that of trypsin-digested holoenzyme.

The m igration of the holoenzyme after trypsin digestion has been previously observed (W arren, 1988). The m igration is similar to that of an enzym e-interm ediate ES2 complex and results from the loss of a 6kDa

fragm ent from the N-term inus of the protein, due to a cleavage after lysine-64. The free apoenzyme appears to have broken dow n on trypsin digestion, although apoenzyme which has reconstituted the holoenzyme is seen to behave as trypsin digested holoenzyme. The results again confirm the less folded nature of apoenzyme in comparison to holoenzyme.