Biology Reference
In-Depth Information
The specific activity (SA) is the activity of an enzyme per mg of total protein.
¼ 10 6 mol converted mg 1 min 1
SA
The SA of a pure enzyme is constant and is a characteristic of the enzyme. Therefore, the
purity of an enzyme at any step in the purification, expressed as%purity, can be determined by:
100
Until purity is achieved, the measured SAwill always be less than that of the pure enzyme
because other proteins besides the desired enzyme are still present in the solution.
In addition to enzyme activity, total protein must be determined at each purification step.
Many methods to accurately quantify total protein are currently in use. The simplest is to
measure solution absorbance at 280 nm. This method is not sensitive and suffers from
many non-protein compounds in the purification buffers that can also absorb at 280 nm.
A revolutionary colorimetric procedure to quantify proteins was published by Oliver
Lowery ( Figure 13.5 )in1951 [10] . For decades this paper was the most cited in all scientific
literature. A more sensitive method to quantify proteins was described by Bradford in 1976
[11] and further improved by Zor and Selinger in 1996 [12] . A Bradford Protein Assay kit,
commercially available from Bio-Rad, is perhaps the most popular protein assay in use
today. Another very popular protein assay is the Pierce BCA (bicinchoninic) Protein
Reagent Assay that is reputed to be the best detergent-compatible protein assay available.
% Purity
¼
measured SA
=
SA of pure enzyme
C. MEMBRANE LIPID ISOLATION
During the early years of biochemistry, knowledge of lipids far exceeded that of
proteins. Isolation of the first protein was not until 1925 when Sumner reported his
work on urease from jack bean [13] . In comparison, the early pioneering lipid extractions
by Chevreul were done in the early 19 th century [14] . Modern membrane polar lipid extrac-
tion procedures had their beginnings with the classic 1957 paper by J. Folch ( Figure 13.6 )
(Folch, Lees, and Stanley [15] ). With slight modifications, this method is still in use today.
Lipid extraction requires that the tissue must be fresh and devoid of lysophospholipids,
oxidized products, large quantities of mono- or di-acyl glycerol, and free fatty acids. The
tissue is homogenized with chloroform/methanol (2:1) at a ratio of 1 g tissue to 20 ml
solvent. The tissue naturally contains a small amount of water that adds to the mixture
but often must be further augmented to improve phase separation. After filtration or
centrifugation to remove solid cellular debris, the liquid mixture is removed and washed
with 0.2 vol of 0.9% NaCl in water. Low speed centrifugation separates the mixture into
two phases. The upper phase, primarily methanol and water, is enriched in very polar
lipids like gangliosides, while the chloroform-rich lower phase contains the phospholipids
and non-polar lipids. This phase is isolated and evaporated under vacuum and further
extractedwithcoldacetone.Theacetoneextraction removes the non-polar lipids leaving
the membrane phospholipids in the acetone-insoluble pellet. Recently, dichloromethane
has occasionally been used in place of chloroform to avoid adverse health effects. Another
very similar procedure involving extraction with chloroform/methanol/water was later
proposed by Bligh and Dyer in 1959 [16] .
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