A colorimetric assay of membrane lipids

Miguel Angel Rodriguez and Liudmila Beletskaya

University of Ottawa, Department of BMI, Ottawa, ON, Canada

1. Abstract
Brilliant-Blue’s visible spectrum in basic aqueous medium is
characterized by a slow quenching and right shifting of its maximum
absorption peak around 560 nm. Membrane vesicles and polar lipids
have been found to largely increase the rate of this effect. By
optimizing the pH and the time of incubation, we have developed a
colorimetric assay for membrane preparations, membrane lipids, and
pure polar lipids in aqueous suspension. The assay is usually linear in
a range of one unit of absorbance and has a sensitivity of about 5 µg
for pure lipids and extracts of membrane lipids and about 0.5 µg for
lipids in membrane vesicles or lipoproteins. Lipids in membranes are
from 10 to 20 times more responsive than lipids alone. Pure
phospholipids and sphingomyelin show a similar level of response
whereas neutral lipids and fatty acids have no effect on the dye’s
absorption. Some soluble proteins show a level of response similar to
the lipids alone, thus the application of the assay to tissue extracts
will require a correction for the contribution by the proteins. This
assay is more specific than the measurement of the absorption at 325
nm due to the light dispersion by the lipid vesicles, and it is about 10
times more sensitive than the latter when applied to membrane
vesicles or lipoproteins.
2. Introduction
• The interaction of Coomassie
BBR
Brilliant Blue R (BBR) with
lipids and membranes results in H3C N
SO3M

the slow decrease of the dye’s

color.
• This effect can be used for the +
HN N CH3
assay of lipids in aqueous
medium.
SO3-
• This colorimetric assay has some OCH2CH3

advantages over the measurement

of the light dispersion at 325 nm.
3. BBR spectrum at pH 13 with time
In a basic medium, the spectrum shows a slow
decrease in absorbance as well as a red shift
4. Effect of lecithin on BBR spectrum
Soy lecithin increases the rate of change of the
BBR spectrum at high pH.
Peak absorbance after 1 h
Rate constant of Abs decrease at pH 11

2
0.3
BBR + lecithin BBR

1.6

0.2
1.2

k (1/min)
Abs

0.8
0.1

0.4

0 0
6 7 8 9 10 11 12 13 14 0 0.2 0.4 0.6 0.8

pH Lecithin (g/L)
5. Optimal conditions of BBR assay
• Buffer: 100 mM phosphate, pH 11
• BBR concentration: 0.05 mg/mL
• Sample: lipid suspension in water
• Volume incubation: 1 mL
• Incubation time: 20 min
• Vortex thoroughly
• Read absorbance at 559 nm
• Read background with lipid alone, no BBR
6. BBR assay of lecithin
• The assay is linear over 1 unit of absorbance
• Lecithin can be used as a convenient standard
Final plots
Absorbance plots
1.2
1.5

A b s d ecrease
A b s 55 9n m

0.8
1

0.5 0.4

0 0
0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8
Lecithin (mg) Lecithin (mg)

BBR + Soy lecithin Soy lecithin Soy lecithin (40%) Egg lecithin (60%)
BBR + Egg lecithin Egg lecithin
7. Assay of lipid extracts from tissues
The response to the assay depends largely on the
source of lipids

Lipid Extracts
1.4
A(559nm) decrease

1.2 Rat liver

1
RL mitochondria
0.8
0.6 Rat heart
0.4
0.2 Pork liver
0
0 0.2 0.4 0.6 0.8

Lipids (mg)
8. Assay of membranes and lipoproteins
The BBR assay can measure cellular membranes
and lipoproteins in the range 0.5 - 100 µg lipids
1.2
A (559n m ) d ecrease

0.8 RL mitochondria

RL microsomes
0.4
AT mitochondria.

0 Lipoproteins
0 50 100
Lipids (ug)
9. Response of pure lipids to the assay
• Polar lipids can be assayed in the range 0.01-1 mg
• Non-polar lipids give no response
1.00
A(559nm) decrease / mg

0.50

0.00

-0.50
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10. Proteins can interfere
Free proteins give a non-negligible response

2.50
A(559nm) decrease / mg

2.00

1.50

1.00

0.50

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11. BBR assay vs. Abs 325nm
The BBR assay is about 10 times more sensitive
when applied to membrane vesicles or lipoproteins
16
BBR assay
Abs 325nm
12
Response (mL/mg)

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12. Conclusions
• The BBR assay can be applied to polar lipids,
liposomes, lipid extracts, cellular membranes
and lipoproteins
• It is usually linear over one unit of absorbance
• It has a sensitivity of 5-10 µg for lipid extracts
and of 0.5-1 µg for lipids in membrane
vesicles or lipoproteins
• Free proteins will cause a background response
• The BBR assay is about 10 times more
sensitive than the A(325nm) measurement of
membrane vesicles and lipoproteins