Protein

Quantitative Determination
of Proteins
In general, there is no completely satisfactory sin-
gle method to determine the concentration of pro-
tein in any given sample. The choice of the method
depends on the nature of the protein, the nature of
the other components in the protein sample, and the
desired speed, accuracy, and sensitivity of assay. Sev-
eral of the methods commonlyused for protein de-
termination are discussed in the sections that follow.
Biuret Test. Compounds containing two or more
peptide bonds (e.g., proteins) take on a character-
istic purple color when treated with dilute copper
sulfate in alkaline solution. The name of the test
comes from the compound biuret, which gives a
typically positive reaction. The color is apparentlycaused by the coordination complex of the copper
atom and four nitrogen atoms, two from each of
two peptide chains (Fig. II-5). The biuret test is
fairly reproducible for any protein, but it requires
relatively large amounts of protein (1 to 20 mg) for
color formation. Because of its low sensitivity, the
biuret assay is no longer widely used.
Folin–Ciocalteu (Lowry)Assay. The quantitative
Folin–Ciocalteu assay (also often called the “Lowry
assay”) can be applied to dried material as well as
to solutions. In addition, the method is sensitive;
samples containing as little as 5 g of protein can
be analyzed readily. The color formed by the
Folin–Ciocalteu reagent is thought to be caused by
the reaction of protein with the alkaline copper inthe reagent (as in the biuret test) and the reduction
of the Cu2 (cupric) ions in the reagent to Cu1
(cuprous) by the tyrosine and tryptophan residues
of proteins. The cuprous ions in turn reduce the
phosphomolybdate–phosphotungstate salts in the
reagent to form an intensely blue complex. Because
the content of these two amino acids varies sub-
stantiallywithin proteins, the color yield per mil-
ligram of protein is not constant. It may differ sub-
stantially from that of a protein standard.
Nevertheless, the method is very useful for follow-
ing changes in protein content, as, for example,
during purification of a protein. This assay is used
in most of the experiments in this book, because it
is convenient and inexpensive.Bicinchoninic Acid (BCA) Assay. The bicin-
choninic acid assay for proteins is based on the same
reactions as the Folin–Ciocalteau assay. Proteins
are again reacted with alkaline cupric ions to form
the biuret complex, and these ions are reduced to
cuprous ions by the aromatic amino acids in the
proteins. In this case, however, the Cu1 ions form
a complex with bicinchoninicacid (Fig. II-6), which
has an intense absorbance maximum at 562 nm.
This assay shows the same variation from protein
to protein as the Folin–Ciocalteau assay, but is
more convenient experimentally and can be made
somewhat more sensitive.
Dye-Binding (Bradford) Assay. The binding of
proteins to Coomassie Brilliant Blue 250 causes a
shift in the absorbance maximumof the dye from
465 nm to an intense band at 595 nm. Determina-
tion of the increase in absorbance at 595 nm as a
function of protein added provides a sensitive assay
Figure II-5 The biuret reaction.
C
NH2
NH2
NH
O
C O
C
NH2
NH2
NH2
NH2
NH NH
O CO
C O CO
2
C
NH
NH
O
C
CHR
O
2
C
NH
NH
OC O
C
CHR
NH
NH
CHR
OC O
or
Cu
2
OH
Cu2 Cu2
or
BiuretsPeptide chains Biuret complexes94 SECTION II Proteins and Enzymology
OOC N
OOC N
OOC N
OOC N
COO N
COO N



Cu1
Cu2
Cu1
Cu1
Protein
Protein–Cu2 complex
Protein–Cu2 complex (biuret reaction)
Uncharacterized protein oxidation products
BCA BCA–Cu1 complex
Figure II-6 Chemistry of the bicinchoninic acid protein assay.
of protein that is quite constant from...