DETECTION AND ANALYSIS OF PROTEINS - A Guide to Methods in the Biomedical Sciences - page 3

its primary structure. The primary amino acid structure dictates local
folding patterns within individual protein domains that are common to
a number of different proteins. These folding patterns form the sec-
ondary structure of a protein. Linus Pauling, who won the Nobel Prize
in Chemistry in 1954 for describing chemicala bonds, used both exper-
imental approaches as well as models of small polypeptide chains to
determine the orientation of the peptide bond. He found that because
of the rigidity of the peptide bonds, amino acids would naturally assume
common secondary structures stabilized by hydrogen bonds. The most
common secondary structures are “beta-pleated sheets” and “alpha-
helices”. The further folding of a protein that gives it its own unique
overall structure is its tertiary structure. The tertiary structure of a pro-
tein is determine and also stabilized by chemical interactions such as
hydrogen bonds, ionic bonds, Van der Waals forces and hydropho-
bic interactions. Further, many proteins form multisubunit complexes,
and the way these interact identifies the quaternary structure of the
protein.
The following sections outline common methods that are used to mon-
itor proteins, analyze their composition and mass, and determine subunit
structure in biomedicine. They also outline various methods used to de-
termine secondary, tertiary and quaternary structures of proteins, and
identify various post-translational modifications.

B. Basic methods for protein analysis

Determination of protein content

Three methods are commonly used to quantify proteins in biological
samples. Two of these are colorimetric assays named after their inven-
tors, Lowry, who developed the first simple method for protein quan-
titation in 1951 (1), and Bradford (2), who developed a more sensitive
colorimetric assay in 1976. The fact that Lowry's paper is one of the most
frequently cited papers in history attests to the importance of methods
to monitor protein concentration.
The Bradford assay is commonly used today due to its sensitivity and
simplicity. It is based on the ability of Coomassie Brilliant blue dye, which
was first used to dye wool, to bind to proteins at acid pH. Binding leads
to a color change, which provides a measure of the amount of total
protein present. The binding of Coomassie Blue is nonspecific and irre-
versible. Most Bradford assays today are quantitated using automated
plate readers at an OD of 595 nm. The amount of protein can be quan-
titated by comparing the absorbance with a standard curve prepared
using a purified protein such as albumin.