La diferencia con los colegios bilingües

As stated in the introduction to this chapter, the aim of the isolation of PAPP-A was to use th at m aterial for N-terminal microsequencing, A scheme (Figure 5.2) th at utilised an affinity of PAPP-A for the amino acid L-arginine combined with steps described by other investigators (Table 1.1, Chapter 1) yielded PAPP-A enriched to approximately 30% of the

proteins present in the final preparation. The only other major protein

As illustrated in the plate inset 2.19 (Chapter 2), PAPP-A could not easily be separated from an unidentified contam inant th at was present as a single chain form with an Mr of approximately 210 - 240 kDa. This contaminating m aterial (hereafter named group-X) was immunodetected using an antiserum directed against normal serum components. It also co­ eluted with PAPP-A in all the methods used to isolate PAPP-A which are described in Chapter 2, section 2.7. The only method th at was successful in removing it, was on a basis of its size by gel filtration. The m aterial

prepared by this scheme was separated from 0C2M by SDS-PAGE under

reducing conditions. It was electroblotted onto a PVDF membrane th at is suitable for use in microsequencing (Plate inset 5.2). The PAPP-A band indicated by ( ★ ) yielded a single N-terminal sequence (Chapter 6, Table 6.2) thus confirming th at the PAPP-A isolated had been separated from other contaminating serum proteins.

The second scheme th at was successful in enriching PAPP-A utilised the described affinity of PAPP-A for heparin and is illustrated in Figure 5.3. The heparin bound fractions (0.3 and 0.6 M) shown in Figure 2.12 (Chapter

2) were subjected to gel filtration (Figure 7.4, Chapter 7) and the fractions

containing PAPP-A were identified as described (Plates 5.1 and 5.2).

The heparin based scheme (Figure 5.3) as illustrated in Plates 5.1 and 5.2 significantly enriched the PAPP-A present and separated PAPP-A from

its main contam inant seen in scheme 1 (Figure 5.2), th at of (X2M. It however

failed to separate PAPP-A from the unidentified X-contaminant. This contam inant has also been observed by Davey et al. (1983) who noted th at it could be removed if a 0.4 M NaCl step was substituted between the 0.3 and 0.6 M NaCl steps during the heparin affinity chromatography stage. This did however result in a considerable reduction in the yield of PAPP-A

and the m aterial was still contaminated with other serum proteins. Sinosich

et al. (1990) removed these by negative affinity immunoadsorption. In this study it was chosen to purify a single chain PAPP-A by elution from a gel slice as described in Chapter 2, section 2.7.12. This method yielded a pure monomeric PAPP-A molecule (Plate inset 5.3).

(G ro u p X) < 205

P la te 5.1

A CBB stained 5% SDS-PAGE gel of fractions from heparin based

purification, Scheme 2.

[Legend: All samples were reduced with 2-mercaptoethanol before separation on this gel. The 0.3 and 0.6 M bound fractions from the heparin affinity column were loaded onto a gel filtration column (see text). The 0.3 M heparin bound fractions separated on the gel filtration column were (1): Fractions 1 - 4. (2): Fractions 5-6. (3): Fractions 7-8. (4): Fractions 11 - 12. (5): The high molecular weight m arkers (Appendix 1) with the arrow indicating the 205 kDa marker. The 0.6 M heparin bound fractions separated on the gel filtration column (see text) were (6): Fractions 1 - 4. (7): Fractions 5 - 8. The PAPP-A band marked was determined by western blotting and subsequent detection with anti-PAPP-A antibodies. The group-X contam inant was also marked.]

1

2

3

( - ) ( + )

( - ) ( + )

<-) ( + )

m ~ m

,

i

\uW

,

\

a

A CBB stained 5% SDS-PAGE gel illustrating the disulphide bridged

structure of the proteins separated by the heparin based purification scheme

[Legend: ( + ) indicated samples were reduced with 2-mercaptoethanol, ( - ) indicated samples were treated with sample buffer th at did not contain 2-mercaptoethanol. The samples separated were from the heparin affinity column and then loaded onto the superDex gel filtration column (see text): (1): 0.3 M bound heparin column, fractions 1 - 4 from gel filtration column. (2): 0.6 M bound heparin column, fractions 1-4 from gel filtration column. (3): 0.6M bound heparin column, fractions 5 - 8 from gel filtration column. (4): The high molecular weight m arkers (Appendix 1) with the arrow indicating the 205 kDa marker. The PAPP-A marked band was determined by western blotting and subsequent detection with anti-PAPP-A antibodies. The group-X contam inant was also marked.]

1B iological S ource M a terial 65 m g/m l

(0.09% PAPP-A)

I

S uperD ex Gel F iltra tio n H e p a rin A ffinity C h ro m ato g rap h y 28/60 % A m m onium S u lp h a te C ut (0.52% PAPP-A) DEAE Io n E x ch an g e C h ro m ato g rap h y (2% PAPP-A) ‘ 90% Yield I

Sam ple c o n c e n tra tio n on C entriprep-100

(P A P P -A )-

F ig u re 5.3

The heparin based purification, scheme 2. P la te In se t 5.3, Pure

monomeric chain PAPP-A.

[Legend: k Biological source m aterial was composed of a pool of late 3rd trim ester plasma and blood lost during delivery at term th at had been treated as described in section 2.7.2, Chapter 2. The plate inset was a CBB stained 5% SDS-PAGE gel as described in section 2.3.2.2.a of the gel-slice eluted m aterial (described in Chapter 2, section 2.7.12) from this purification scheme was illustrated in Plate 5.1. This preparation yielded a pure monomeric chain. PAPP-A. Note: (1): Represented eluted pure monomeric PAPP-A. (2): Represented high molecular weight m arkers (Appendix 1) with size illustrated in kDa.]

5.3.2 A Schem e T h at Sig n ifican tly E n ric h e d th e U n d efin ed (G roup-

X) C o n tam in an t

Sinosich (1988) using CAIE with the dye, Red-120 observed th at the

PAPP-A immunoprecipitate was removed if this dye was included in an intermediate gel, suggesting that it might be possible to isolate PAPP-A by dye affinity chromatography. A scheme incorporating this dye was devised (Figure 5.4).

Red-120 Dye Affinity C h ro m ato g rap h y C ib ach ro n B lue Dye A ffinity C h ro m ato g rap h y

28/60 % A m m onium

S u lp h a te C ut (0.52% PAPP-A)

bio lo g ic a l S o u rce M aterial 65 m g/m l (0.09% PAPP-A) DEAE Ion E x ch an g e C h ro m ato g rap h y (2% PAPP-A) 90% Yield L -A rginine A ffinity C h ro m ato g rap h y F ig u re 5.4

Enrichment scheme for group-X contaminant. (P late In se t 5.4 represented

electroblotted m aterial purified using this scheme.)

[Legend: h Biological source m aterial was composed of a pool of late 3rd trim ester plasma and blood lost during delivery at term that had been treated as described in section 2.7.2, Chapter 2. The steps used in this purification were carried out as described in Chapter 2, section 2.7. The m aterial in the final step was reduced with 2-mercaptoethanol and separated on a 5% SDS-PAGE gel, it was electroblotted on a PVDF membrane as previously described in Chapter 2, sections 2.3.2.2c and 2.4.1 and stained with CBB. Note:

The arrow indicated molecular weight m arker with the size in kDa. The W estern blotted m arked band (Group -X) did not demonstrate an affinity for anti-PAPP-A antibodies using the conditions previously described. The protein present in this band was sequenced by Dr A. Moir (Appendix 3).]

Surprisingly the Red-120 dye did not enrich the PAPP-A component but the group-X contaminant. This was an uncharacterised group of proteins, which in N-terminal microsequencing generated more than one N-terminal amino acid. The amino acids present did not correspond to the N-terminal sequence th at had been obtained for PAPP-A prepared by the purification

scheme 1 (Chapter 6). That the CAIE technique yields different results to

th at obtained by chromatography was also seen when the LEL lectin was

used for purification. A further analysis of the implications of this contaminating group is given in Chapter 7.

The PAPP-A isolated and defined by the schemes described in this chapter produced a monomeric form of PAPP-A th at was free of pro-MBP and yielded a single N-terminus (Chapter 6, Table 6.3). This m aterial was thus suitable both as a substrate for limited chemical/proteolytic cleavage to obtain further prim ary sequence information (Chapter 6) and provided a source of pure monomeric PAPP-A th at was used to study some of the physico-chemical properties of the PAPP-A molecule (Chapter 7).

CHAPTER

SIX

Chapter Six