Purification o f serum C

C3 was purified from plasm a essentially by the m ethod according to Dodds,

1993. 5 ml hum an plasm a containing 5 mM EDTA, 0.5 m M Pefabloc SC, was made 5%

(w/v) w ith polyethylene glycol (PEG M W 3350, Sigma) by adding 2.5 ml o f a 15%

(w/v) solution o f PEG in buffer A (20 mM Tris, 50 mM s-am inocaproic acid (EACA),

5 mM EDTA, 0.02% (w/v) NaN^, 0.5 mM Pefabloc SC, pH 7.5 w ith HCl). The mixture

is stirred gently at 4°C for 30 m in on a roller-m ixer and spun at 10,000 r.p.m. for 20 min

to remove the resulting precipitate. The supernatant was loaded on a Q-Sepharose 26/30

Fast Flow colum n (Amersham Biosciences, Amersham ) at a flow rate o f 2 m l/m in

equilbrated w ith 95% buffer A and 5% buffer B (IM N aCl) and eluted w ith a 200 ml

linear salt gradient to a final buffer containing 500 mM NaCl. The resulting elution

profile is shown in Figure 5.3a). The supernatant was passed through the colum n at room tem perature but the fractions were collected into tubes on ice.

C3 is eluted after a protein o f blue colouration, ceruloplasmin, approximately halfway through the gradient. The pool containing C3 is diluted w ith h a lf the volume

with de-ionised w ater to lower the ionic strength sufficiently to allow binding the

M onoQ H R 5/5 column (Amersham Biosciences, Amersham). The diluted sample was run in two or three separate batches to avoid overloading the colum n and reducing

resolution. The M ono Q 5/5 column is equilibrated with 90% buffer A, 10% buffer B

(IM NaCl). The C3 containing sample is loaded and imm ediately eluted w ith a 20 ml

linear gradient to 300 mM N aCl at a flow rate o f 1 ml/min. The resulting elution profile

is shown in Figure 5.3b). The C3 fractions were dialysed in PBS (Sigm a D-5652, 137 mM N aCl, 2.7 mM KCl, 8.1 mM Na^HPO^, 1.5 mM KH^PO^, 0.5 mM EDTA, 0.02%

NaNg (w/v) , Im M Pefabloc SC, pH 7.5) prior to concentration using a Centiprep-10

concentrator (MW cut-off at 10,000 Da, Amicon). The concentration o f C3 was

determined from an absorption coefficient 9.7 calculated using SLUV from its primary

sequence (1%, 280 nm, 1 cm path length; Perkins 1986). The prim ary protein amino acid

sequences for C3 (C 0 3 HUM AN) was extracted from the SW ISS-PROT database at

EM BL through the HGM P-RC using protein accession num ber P01024. The signal

a)

1000 800

§

0.6 200 0.2 280 160 240 80 120 200 40 Vol (ml) b) e 0.8. _•400 CO 0.6 _•300 200 100 L.. 1—4 vet (ml)

F ig u re 5.3. a) The elution profile obtained when 5ml o f hum an ED TA plasm a is loaded onto a 26/30 column o f Q-Sepharose and eluted w ith a 200m l linear gradient from 50 to 500 mM NaCl. The absorbance at 280 is indicated by the solid line and the m olarity o f the N aCl is shown by the dashed line. The regions o f C3 and C4 elution are depicted in the diagram. C3 is eluted after ceruloplasmin, a protein o f blue colouration, approxim ately halfway through the gradient. C3 devoid o f any contam ination o f ceruloplasm in is taken from the fraction depicted C3II.

b) The elution profile obtained when one part in three o f the fraction from a) (C3I, C3II) is loaded onto a M onoQ H R 5/5 colum n and eluted w ith a 100-300 mM N aCl gradient. The absorbance at 280 is indicated by the solid line, and the molarity o f

5.4.2.3. Factor I-dependent cofactor activity o f FH A ssay

The activities o f FI and FH were measured by incubating the serum proteins with

C3(NHg), generated by the thiolester cleavage o f C3 using ammonia. C3 was converted

to C

3

(NH

3

) by dialysis into 100 mM ammonium bicarbonate at pH 8.0 and incubation

at 37°C for 1 hr. FI cleaves amidated C3, [C

3

(NH

3

)] w hich was used as an equivalent

o f C3b, essentially as described in Sim and Sim (1983). Factor I-dependent cofactor

activity o f FH was assayed by m ixing 5 pg o f C3b, 0.2 pg o f FI and 0.1 pg o f FH in a

20 pi volum e and incubating for 15 m in at 37 °C. The sam ples w ere reduced and

analysed on SDS-PAGE (Figure 5.4). The breakdown o f the a ' chain o f C

3

(NH

3

) with

a M W o f 108,000 into two fragments o f M W 68,000 and 43,000 was visualised by

Coomassie blue staining o f SDS-PAGE.

5.4.3. Sm all angle solution scattering

5 4.3.1. X -ray data from Station 2.1 at the Synchrotron Radiation Source

X -ray scattering data acquisition was perform ed in five independent sessions at

the Synchrotron Radiation Source at Daresbury, W arrington, UK at the solution

scattering cam era at Station 2.1 equipped with a 500-channel quadrant detector (Towns-

A ndrews et a l , 1989; W organ et a l , 1990). Experim ents were perform ed with beam

currents in a range o f 111-230 mA and a ring energy o f 2.0 GeV. Sample-to-detector

distances o f 3.33 m to 5.64 m were used, which yielded a m axim um Q range o f 0.05 -

2.2 nm'^. The Q range was calibrated using fresh, wet, slightly stretched rat tail collagen,

based on a diffraction spacing o f 67.0 nm. FH concentrations betw een 0.1 to 16.0 mg/ml

were measured at 15°C in Perspex cells o f sample volume 20 pi, contained w ithin mica

windows o f thickness between 10 to 15 pm. A cquisition tim es were 10 m inutes using y

10

time frames which were individually checked for radiation damage, and buffers and ^

samples were measured in alternation to minimise background subtraction errors. Runs

were normalised using an ion chamber m onitor positioned im m ediately after the sample.

D ata reduction was performed using the standard Daresbury software package OTOKO

(Boulin et a l , 1986). The buffer and sample runs were corrected by dividing by a

detector response measured for at least

8

h using a uniform ^^Fe radioactive source. R educed curves were calculated by subtraction o f the buffer runs from those o f the

FH

FH

In document Structural studies of SCR domains in multidomain complement proteins (Page 162-165)