4. A COMPARISON OF THE STRUCTURE AND STABILITY OF THE
4.2.5 Urea PAGE
Urea PAGE was performed as previously described (Makey et a i, 1976) using a 6%
polyacrylamide gel that contained 6 M urea. The mobile phase consisted of 100 mM
Tris, 10 mM boric acid and 1.6 mM EDTA pH 8.4. Electrophoresis was carried out at 90-100 V/20 mA for 16 hours. Staining and destaining was carried out as described in Section 3.2.2.
Using this technique it is possible to identify the binding site of transferrin in which iron is located. The following bands can be discriminated (according to increasing mobility); Tf (iron-free protein), Tf-Fe^ (monoferric transferrin, iron bound to C-lobe), Fe^-Tf (monoferric transferrin, iron bound to N-lobe) and Fe^-Tf-Fe^ (diferric transferrin, iron bound to both lobes).
4.2.6 FTIR spectroscopy
FTIR spectra were recorded on a Ni col et 740 FTIR spectrometer interfaced to a 680 spectral workstation. For samples in H^O, 500 scans were signal averaged. The spectrometer was continuously purged with dry air (supplied by a Balston 75-10 air dryer) to minimise contributions from water vapour in the spectral region of interest. A sample shuttle was used to allow background and sample spectra to be recorded alternatively (shuttling every 50 scans). Single-sided interferograms were recorded and apodised with a Happ-Genzel function prior to Fourier transformation to give a resolution of 2 cm '\ The specialist 6pm pathlength cell described in Section 2.6.4. 8 was
used for samples in H2O. Buffer spectra were recorded under conditions identical to
those used for protein spectra. Temperature control was achieved by placing the sample cell in a cell jacket supplied with circulating water. The circulating water was maintained at the desired temperature using a Haake thermostatted water bath. Where temperature was varied (dénaturation runs), the bath temperature was controlled by a Haake PG-40 temperature programmer, and samples were heated over a linear temperature gradient from 20°C to 80°C and then cooled back to 20°C. A complete run lasted »27 hours. Spectra were recorded continuously during this time period and sets of 500 spectra were coadded. This process yielded 61 averaged spectra with a temperature interval of approximately 2°C.
All the spectra were transferred to a personal computer for data analysis. Buffer subtraction was carried out automatically using the procedure outlined in Section 2 .6.4.7.
Ni col et PCER software was used to generate deconvolved (Kauppinen et a/. 1981) and second-derivative spectra. All second-derivative spectra presented in this work were calculated using a 13 cm'^ data window (Savitsky-Golay smoothing). Quantification of FTIR spectra was performed using factor analysis (Lee et a l, 1990).
4.2.7 DSC
DSC data was recorded on a Polymer Laboratories DSC Gold system interfaced to an IBM PS/2 personal computer. See Section 3.2.4 for further details.
4.3 Results
4.3.1 Specific iron loading into the C-lobe of human serum transferrin
Figure 4. 1 shows the urea page profile of human serum transferrin partially saturated
with iron using Fe NTA. It can clearly be seen that the iron binds primarily to the C lobe when it is presented in the form of Fe NTA. Samples of 45% and 60% iron- saturated transferrin were used to study the thermal stability of the iron-free N-lobe.
Fe,-Tf-Fe, Apo Tf Fe,-Tf
Figure 4.1: Urea PAGE analysis of human serum transferrin partially iron-loaded with Fe NTA. Lane 1) apo-transferrin. Lane 2) 15% saturated. Lane 3) 30% saturated. Lane 4) 45% saturated. Lane 5) 60% saturated. Lane 6) 75% saturated. Lane 7) 90% saturated.
4.3.2 Purification of the isolated C-lobe of human serum transferrin
Figure 4.2. shows the HPLC trace obtained from trypsin digested HST eluted from a DEAE Sepharose column. After HPLC, samples with retention times of 13.5-15 minutes were collected and pooled. SDS PAGE analysis of these fractions is shown in Figure 4.3. A single band with approximate molecular weight 43,000 Da is obtained.
4.3.3 Purification of the isolated N-lobe of human serum transferrin
SDS PAGE analysis of isolated N-lobe of HST used for structural studies is shown in Figure 4.4. A single band with approximate molecular weight 36,000 Da can be observed.
CO û
s
CO CO CO Û Û Û CO o CO CM. ▼ 'r 1f '1 0 0 i
90-
80-
70-
60-
Q L L50-
^ 40-
0 0 CNJ^ 30-
2 0
-10
-0
5
10
15
20
25
30
R e te n tio n T im e (min)
Figure 4.2: Typical HPLC profile obtained from typsin digested HST eluted from a DEAE Sepharose column
200kDa 97kDa 69kDa 46kDa
30kDa
Figure 4.3: SDS PAGE analysis of the purified isolated C-lobe of human serum transferrin. Lane 1) 5 pg protein after HPLC. Lane 2) 2.5 pg protein after HPLC Lane
3)5 pg protein before HPLC
80kDa
36kDa
Figure 4.4: SDS PAGE analysis of the purified isolated N-lobe of human serum transferrin Lane 1) 7 pg protein Lane 2) 7 pg protein. Lane 3) 8 pg protein. Lane 4)
3 pg protein. Lane 5) 1 pg protein
4.3.4 Secondary structure comparison of isolated N- and C-lobes in their apo- form The second-derivative spectra of isolated C- and N-lobes of HST in H2O are shown in
Figure 4.5. Bands at 1657 cm'^ are assigned to a-helix/random coil structures whilst those at 1688 cm'^ and 1632 cm‘‘ are assigned to P-sheet. Components at 1679-1673 cm'^ are assigned to turns. A shoulder observed at 1649 cm*^ in the spectrum of the isolated C-lobe is assigned to random coil structures. The amide II band is observed at 1549 cm'^ and the component at 1517 cm'^ is assigned to tyrosine sidechains. The amide I bands of the absorption spectra of the isolated N- and C- lobes of HST were analysed for secondary structure content by means of factor analysis (Lee et a l, 1990) and the results are shown in Table 4.1. For comparison the secondary structure of intact human apo- transferrin estimated by FTIR spectroscopy is also shown.
The quantitative data show that the secondary structure content of isolated N- and C- lobes are very similar.
a-helix P-sheet Turns Undefined
C-lobe 47 % 25 % 1 0 % 18 %
N-lobe 53 % 23 % 1 0 % 14 %
Intact 52% 23% 7% 18%
Table 4.1: Secondary structure analysis of isolated C- and N-lobes of human serum transferrin
C-lobe
o> 00 (O 00 lO o>\>
r o m lO CD N - l o b e (%) lO G ) lO 1600 1550 1500 1800 1750 1700 1650Wavenumber (cm^ )
Figure 4.5: Second-derivative FTIR spectra of the isolated C- and N-lobes of human serum transferrin. Spectra were recorded at a protein concentration of approximately 40 mg/ml in a HjO buffer containing 20 mM HEPES, 0.154 M NaCl pH 7.4