CAPÍTULO 7: RESULTADOS Y DISCUSIÓN
7.5 PERFILES DE TEMPERATURA DEL VIDRIO
2.4.3 . 1 Immobilisation of trypsin of Sepharose CL-6B Trypsin was covalently attached to Sepharose beads as outlined earlier
(2.3.4.3. 1 ) . The trypsin activity associated with the resin and with the
washings from the resin was measured by the hydrolysis of the chromogenic substrate BAPNA as described in 2.3.4.3 . 1 . The data is presented in table 2 . 4 .
Table 2 .4 Trypsin activity measured by the rate of hydrolysis of the chromogenic substrate BAPNA .
Background rate of Rate of BAPNA Sample BAPNA hydrolysis hydrolysis after
(�A 405 .ml-l.min-1) addition of sample (�A 405.ml-l·min-l)
Trypsin-Sepharose 0.00 0.79
Washings from trypsin- 0.00 0.00
S arose
The data in table 2.4 indicates that trypsin had been immobilised on
Sepharose and maintained its catalytic activity. The absence of trypsin in the washings from the resin indicates complete or near complete attachment of the ligand to the Sepharose support. Having ascertained that the enzyme
immobilised on the resin was active, the trypsin-Sepharose was used to partially digest samples of iron saturated bovine lactoferrin.
2.4.3.2 Digestion of lactoferrin with immobilised trypsin and the isolation of the 50 kDa C-terminal peptide
Iron loaded bovine lactoferrin was partially digested with immobilised trypsin as outlined in 2.3.4.3.2. The progress of hydrolysis of lactoferrin was
followed by gel electrophoresis. Figure 2.6 illustrates a typical
electrophoretic pattern of bovine lactoferrin digested with immobilised trypsin. After 9.5 hours of gentle mixing at room temperature the 80 kDa lactoferrin band was almost completely converted into 50 kDa and 30 kDa polypeptides. The peptides were separated by preparative gel electrophoresis
on 10% (w/v) polyacrylamide-SDS gels. A fluroscein isothiocyanate labelled sample of the partially digested lactoferrin was loaded into a marker track on the side of the preparative gel. The position of the peptides after
electrophoresis was determined by examining the marker track under ultraviolet light. Figure 2.7 A shows a marker track from a preparative gel exposed to ultraviolet light. The fluorescent labelled peptides were clearly visible when exposed to ultraviolet light on a transilluminator. Small sections of preparative gel corresponding to each of the three major components (80, 50, and 30 kDa bands) were cut from the gel. These sections were placed in the wells of an analytical 10% (w/v) polyacrylamide-SDS gel and subjected electrophoresis to determine the molecular weight of the peptides. Figure 2.7 B illustrates the typical electrophoretic pattern from the preparative gel
sections. Having determined that the fluorescent markers indicated the presence of the three polypeptides in the gel, the 50 kDa peptide was isolated from the preparative gel by electroelution. The 50 kDa fragment of lactoferrin was eluted into a dialysis bag, dialysed against 0.01 M NH4HC03 and lyophilised. The lyophilised material was resuspended in sterile dH20. Figure 2.8 illustrates a silver stained 10% (w/v) polyacrylamide-SDS PHAST gel of the 50 kDa polypeptide isolated from the preparative gel. The major component in the electroeluted sample is an intensely stained band which migrates to the same position as the 50 kDa C-terminal tryptic peptide of bovine lactoferrin.
1 2 3 4 5 6
Gel details: 10-15% (w/v) polyacrylamide-SDS PHAST gel stained with Coomassie Brilliant Blue.
Loading details:
1 and 3. Bovine lactoferrin digested with immobilised trypsin for 9.5 hours.
2 and 6. Molecular weight markers (see figure 2.4 for details). 4. Bovine lactoferrin digested with immobilised trypsin for 1.0 hour.
5 . Bovine lactoferrin prior to digestion with trypsin.
Figure 2 .6 PHAST gel electrophoresis of bovine lactoferrin before and after limited proteolysis with immobilised trypsin.
A. B.
1 2 3 4 5
...-..--Uncut Lf
...-..--50 kDa
...-..-- 30 kDa
Figure 2 . 7 Analysis o/partially digested lacto/errin by gel electroplwresis. A. Gel details: 1 0% (w/v) polyacrylamide-SDS preparative gel.
Loading details: Bovine lactoferrin partially digested with immobilised trypsin and stained with FITC and photographed on a Transilluminator. B. Gel details: 10% (w/v) polyacrylamide-SDS analytical gel stained with Coomassie Brilliant Blue.
Loading details: 1 . Molecular weight markers (see figure 2.4 for details). 2. 30 kDa lactoferrin tryptic peptide excised from the preparative gel.
3. 50 kDa lactoferrin tryptic peptide excised from the preparative gel. 4. Uncut bovine lactoferrin excised from the preparative gel.
5. Bovine lactoferrin partially digested with immobilised trypsin.
1 2
�Uncut bovine lactofenin � 50 kDa tryptic peptide
� 30 kDa tryptic peptide
Gel details: 10-15% (w/v) polyacrylamide-SDS PHAST gel stained with silver nitrate.
Loading details:
1. Electroeluted 50 kDa bovine lactoferrin tryptic peptide isolated from a preparative polyacrylamide gel.
2. Bovine lactoferrin partially digested with immobilised trypsin.
Figure 2.8 Polyacrylamide-SDS gel electrophoresis of the C-terminal 50 kDa tryptic peptide of bovine lactoferrin isolated by preparative gel
Approximately 100 �g of purified 50 kDa peptide in 50% fonnic acid was subjected to automated N-tenninal sequencing as described in 2.3.4.3.3. The N-tenninal sequence data from the 50 kDa peptide is presented in table 2.5. Table 2 .5 The N-terminal amino acid sequence of the 50 kDa tryptic pep tides from bovine lactoferrin. The sequence of the corresponding region of human lactoferrin is listed above the bovine lactoferrin peptides. The boxes indicate the residues that are identical in all the members of the transferrin family. See appendix III for the alignment of the complete bovine lactoferrtn amino acid sequence with other members of the transferrinfamity.
Human Lf . , GKDK S P 50 kD Bovine Lf Peptide A. 50 kD Bovine Lf Peptide B. S R 2 90 3 0 0 3 1 0 SAI GF S RVP P R I D S G . . SALGFL R I P S KVD . . SAL GF L R I P S KVD . .
Two peptides were sequenced simultaneously. These two peptides were present in virtually equimolar amounts in the trypsin digested lactoferrin. The sequence of the two pep tides was identical but staggered by two cycles. The most likely explanation for this observation is cleavage at one of two basic residues separated by a single amino acid (serine) residue. Either of these basic amino acids could have been recognised as the site of hydrolysis by trypsin. The sequence of the N-tenninal of the 50 kDa peptide from bovine lactoferrin was compared to the entire human lactoferrin sequence (Metz Boutigue et al., 1984). The N-tenninal of the 50 kDa bovine lactoferrin peptide aligned to position 282 in the human lactoferrin sequence. The bovine and human sequences share approximately 75% identity over the 33 amino acids sequenced. Examination of the tertiary structure of human lactoferrin (Prof. E.N. Baker, personal communication) showed that this sequence corresponds to an external loop on the N-tenninal lobe of the protein. It is reasonable to assume, due to the high degree of sequence homology in transferrin family, that the gross tertiary structures of human and bovine lactoferrins will be similar. This has been supported by recent X ray crystallographic studies on bovine lactoferrin which suggest that the tertiary s tructure of human and bovine lactoferrin are similar (Prof. E.N. Baker and Dr. B.F. Anderson, personal communication). Further, it is also reasonable to assume that the basic residues on this external loop fonn a hypersensitive site for trypsin degradation. Incubation of iron-loaded human
lactoferrin in the presence of trypsin also results in the hydrolysis of the protein to give two large (50 kDa and 30 kDa) peptides (Brock et ai., 1 97 6; Brines and Brock, 1 983).
2.4.3.3 Tryptic mapping and sequencing of selected peptides from bovine l actoferrin
Bovine lactoferrin was carboxylmethylated, reacted with maleic anhydride and then digested with trypsin. The resultant acid-soluble peptides were separated by reverse phase HPLC as described in 2.3.4.3.4. Figure 2.9 shows the tryptic map generated by the separation of the peptides on a C-1 8 Vydac column. Several column fractions were sequenced by automated gas phase sequencing. Table 2.6 shows the amino acid sequence data obtained from the selected column fractions that were sequenced.
Table 2 .6 The amino acid sequence of selected acid soluble tryptic peptides from carboxymethylated,maleonated bovine lactoferrin separated by reverse
phase HPLC.
Fraction number Sequence
3 KVNR 4 YIR 7 KVNR and EDFR 8 KSCHTGLGR 1 1 KPVTEAQSCHLAVGPNHAVVSR 1 2 KPVTEAQSCHLAVGPNHAVVSR 14 WQWR 1 5 APVDAFKECHLAQVPSHAVVAR 1 7 APVDAFKECHLAQVPSHAVVAR
1 8 YLTTLKNLR and AFALECLTR
Fractions 7 and 1 8 contained two discrete pep tides which sequenced
simultaneously. The predominant sequences from fractions 1 1 and 1 2 were identical as were the sequences from fractions 1 5 and 17. The presence of
HPLC elution profile of tryptic pep tides from bovine lactoferrin 100 80
§
0 """'"M �
60 N · ... ... --g ro d) d) I-< U§
40 .D ..c: a U � "--" .D -< 20 0 1 2 0 10 3 4 7 20 Column: C- 18 Vydac HPLC apparatus: Spectra PhysicsGradient: 5 minutes at 100% buffer A.
90 minutes at 100% buffer A to 100% buffer B.
17 18 /."'�/.�/-'/-//"
:
i
13 14 •.•..••..•..••.. 20 22 23 j:Q •. ···16 19 21 � 15 20 30 40 50 60 Time (minutes)Absorbance range: 0-2.0 Chart Speed: 20 cm/hr
Buffer A= 0. 1 % TFA in H20
Buffer B= 0. 1 % TFA in H20/isopropanol/acetonitrile (1/1/1 (v/v/v)). 250 ill of acid (0.1 % TF A in H20) soluble tryptic peptides from bovine lactoferrin treated with maleic anhydride. (Approximately 5 mg of peptides). Notes: Numbers indicate fractions collected manually. The dotted line represents the buffer gradient.
Figure 2.9 Tryptic map of bovine lactoferrin after incubation with iodoacetic acid and maleic anhydride. 00
the same pep tides in two separate fractions was probably due to the peptides associating with minor peptide components in the sample. Most of the peptide sequences obtained could be aligned to the human lactoferrin amino acid sequence. Several of the shorter peptides, namely 3,4,7 and 1 4, could only be placed once the entire amino acid sequence for bovine lactoferrin had been deduced from the messenger RNA sequence (see Chapter 3).
2.4.4