Heparin-Sepharose affinity chromatography

INTRODUCTION

CHAPTER 2 CHAPTER 2

2.1. Laboratory procedures 1. Subjects

2.1.9. Heparin-Sepharose affinity chromatography

VLDLI and YLDL2

were subfractionated

by

heparin-Sepharose affinity chromatography (Shelburne

and Quarfordt, 1977) into two

populations of

particles; a heparin-bound (bound) and a

heparin-unbound ^(unbound)

fraction. Heparin

Sepharose

CL-68

(#17 -0467

-Ol> was ^obtained

^from

Pharmacia (Pharmacia

Fine

Chemicals, Uppsala, ^Sweden).

Heparin

affinity

columns (1.0 x 30.0 cD, Bio-Rad

Econo-column,

Bio-Rad

Laboratories, Richmond)

were

equilibrated

with 0.05 M

NaCl/2

mM

PO¿

pH 7.4

^(starting buffer). The PO4 solution was prepared by titrating 0.5

M

NaH2PO4.2H2O ^(BDH Chemicals, Melb., Aust.) and 0.5

M

Na2HPOa.l2H2O

(Ajax

Chemicals, Sydney,

Aust.).

Samples containing between

2-3 mg of

protein were dialysed against

the

starting buffer.

The

unbound fraction was eluted,

with the

starting buffer,

at a flow

rate

of

2O

ml/hr at 4oC, and

collected

in 2 ml

fractions. After approximately

2

hours when

the

optical density

of the

fractions, measured at

280 nm, was zeÍo

a 0.8 M

NaCl/2mM PO4 buffer was ^used

to

elute the ^bound fraction.

All

fractions were measured

at

280

nm

and pooled

into two

groups.

Measures

of total ^protein

showed recoveries generally greater

than

^85Vo.

Although diluted after being ^separated

on the

column

the

concentration

of

^the

unbound

and bound pooled

^samples

was sufficient to permit apo B

^and

triglyceride

concentration

and radioactivity

determinations.

2.L.10. Lipoprotein

delipidation

Preparations

of lipoproteins

\Ã/ere

delipidated using the

^methanol,

chloroform,

diethyl ether (2:l:9), (vlvlv)

method

of

Herbert

et al

^(1973).

Methanol, chloroform

and diethyl ether were

obtained

from BDH

Chemicals

(Melb., Aust).

At

each step

of

the procedure samples were placed

at

-10oC for a

period of 12 ^hours or more to

improve delipidation

efficiency and

^hence

protein

recovery. Samples

were

centrifuged

to petlet

apolipoproteins

and

^to

enable removal of non ^protein

phase. Some

lipid rich

^{samples required}

additional delipidation

to

separate

lipid from

protein. Following removal

of

the

lipid

^{phase protein samples were dried under}

a

stream

of

^nitrogen.

2.l.Ll. Quantification of apolipoprotein

^B

Apolipoprotein

B for

quantitation

and specific activity

determinations

was precipitated

from

lipoprotein samples using

a

modification

of the

method

of

^Egusa

et al

^(1983). Comparison

of this and the

tetramethylurea ^(TMU)

method for

determining

apo B

concentration

have

shown

no

differences,

except that the

isopropanol method

is simpler and less time

^consuming

(Holmquist

et

â1, 1975). Concentrated protein samples were diluted

with

saline

and solutions

with high

salt content were dialysed against Na2 EDTA-saline ^(d.

1.006 g/ml 0.1M EDTA, pH 7.4). Between 500-700 pg of ^protein, in about

I

ml, was

mixed

with

an equal volume

of

1007o isopropanol (BDH Chemicals, Melb., Aust.).

After

vigorous

mixing

samples were incubated

at

room temperature overnight and centrifuged

at

1000

x g for 30

^minutes. The supernatant was aspirated and

the pellet

washed

with a volume of l00Vo

isopropanol.

The pellet

was

recentrifuged

and the

supernatant removed. Samples

were air dried

^and

delipidated, using

the

method described

in

Section 2.1.10.

This

delipidation ^step was found be

to

^essential when using lipemic samples

in

order

to

^{remove lipid}

labeled radioactivity. After delipidation samples were centrifuged,

^the

supernatant removed,

and the

pellets

air dried.

Aliquots

of 1N

NaOH ^(Ajax Chemicals, Sydney,

Aust.) were

added

to the ^pellet and the

solution was

incubated at

^40o

C until the pellet ^was

dissolved.

Protein content

was determined using

the

procedure described

in

Section 2.1.2.1.

2.l.Ll.l. Preparation of

antisera

Apo B

was isolated

from a

^washed

LDL

fraction

(d.

1.030-1.040 ^g/ml) (Reardon

et al

1981),

and

^protein concentration determined.

This

narrow-cut

of LDL

was used

as the

standard

in the

assay procedure.

An

aliquot

of

^the

lipoprotein sample containing

1

mg

of

protein was mixed

with 1 ml of

^Freund's

adjuvant (Commonwealth Serum

Labs., Melb., Aust.) and mixed using

the

double syringe

technique.

The

antigen

mixture was injected into

multiple subcutaneous sites

in a rabbit.

Afrcr

2-3

weeks

a

booster injection \vas ^given.

The rabbit was bled after

antisera

had

reached

a sufficiently high

titer.

Antisera

was

tested

for

monospecificity

to apo B on

immunodiffusion ^gels

against other plasma proteins and apoproteins using the ^method

^of

Ouchterlony ^(1958).

2.1.11.2.

Immunoassays

for apotipoprotein B

concentration

Immunoassays

for the

determination

of apo B

concentration were

carried out by the rocket

immunoelectrophoretic procedure described by Reardon

et al

^(1981).

This

method uses Lipase-TG

(EC

3.1.1.3) (Calbiochemm

Corp.,

La Jolla) to

^hydrolyse triacylglycerol

in

lipoprotein particles. Several

workers (Schonfeld

et al,

^{7979: Albers}

et

â1, 1980) have demonstrated that lipid

in triglyceride-rich

lipoproteins

can mask the

immunoreactivity

of the

^B

apoprotein.

Due to the

considerable heterogeneity

in lipid

composition ^across

the

lipoprotein spectrum

it is

important

to

ensure

that lipid

does

not

interfere

with the

measurement

of the

apoB. The effect

of the

^lipase

is to

convert all standards and

test

lipoprotein particles

to LDL-like

particlos,

this

ensures that

the

standard and

test

samples are immunochemically identical.

This

method of measuring apo

B

has been compared

to that of

^Kane

et al

⁽¹⁹⁷⁵⁾ using the

1,1,3,3-tetramethylurea

(TMU)

procedure.

Values

obtained

for the

^lipase-

incubated samples closely approximated those

of the TMU

method ^{(Reardon et} al,1981).

Gels (1.0 mm in

thickness)

of lVo (w/v)

agarose

B

(Pharmacia Fine

Chemicals, Uppsala, Sweden)

in Barbital buffer II

^(Bio-Rad Laboratories,

Richmond),

pH 8.6, and

containing

l-27o (w/v)

antiserum \ryere ^prepared.

Electrophoresis was carried

out for

16

hr at a field

strength

of 8

V/cm. Gels

were stained,

with

Coomasie Blue Brilliant R-250, according

to

^{Laurell (1972).}