40 BIOCHEMISTRY FUNDAMENTALS
Initial N - terminal and C - terminal sequence determination may be made using
end - group analysis. Addition of the reagent fl uorodinitrobenzene (FDNB), for
instance, reacts with free amine to form a dinitrophenyl derivative used to
identify the N - terminal amino acid. Carboxypeptidase will remove the C -
terminal amino acid. Closed - end (circular) peptides or those with modifi ed
N - terminal or C - terminal amino or carboxyl groups will not be detected. The
Edman degradation is a method for removing the N - terminal amino acid. The
process may be repeated successively to sequence an entire peptide. Polypep-
tides may be broken down into fragments by cleaving at specifi c acids. Trypsin,
for instance, will cleave residues with positively charged R groups (lys and arg)
on their carboxyl side, and chymotrypsin will cleave residues with aromatic
(phe, tyr, trp) or bulky aliphatic (ile, val) R groups. CNBr cleaves at the
carboxyl side of the methionine peptide bond. End - group analysis, Edman
degradations, and enzyme treatments may be repeated on different protein
fragments to determine sub - sequences and create “ overlappings. ” Large
peptide fragments are positioned relative to one another after a second or
third treatment creates other fragments whose sequences extend across
the initial cleavage points. Eventually the original polypeptide sequence is
reconstructed.
Many other methods are used in the analysis of protein mixtures or purifi ed
proteins. Ultracentrifugation separates proteins according to size and can
determine their molecular weight. Gel fi ltration (or size exclusion) chroma-
tography separates proteins according to size. Elution time of the protein
molecules through a column is related to molecular size, with the largest pro-
teins eluting fi rst and the smallest last. Ion exchange chromatography sepa-
rates proteins based on their charge, whereas affi nity chromatography separates
proteins that bind specifi cally with certain chemical groups. Electrophoresis
separates different proteins based on their net charge and based on mass if
they have the same charge. Isoelectric focusing separates proteins based on
their isoelectric points — the pH at which the individual amino acids exist as
zwitterions. More information on these methods can be found in reference 7.
The three - dimensional structure of a protein — its secondary, tertiary, and qua-
ternary structure — has been determined for hundreds of proteins (and thou-
sands of protein variations) using X - ray crystallography in the single - crystal
solid state and nuclear magnetic resonance (NMR) spectroscopy in solution.
These techniques are discussed in detail in Chapter 3. The amount and type
of secondary structure in a protein may be determined by circular dichroism
(CD) and magnetic circular dichroism (MCD) spectroscopy. A detailed
description of CD and MCD spectroscopy applied to bioinorganic molecules
is found in Chapter 5 of reference 8. Mass spectrometry (MS) can determine
the structure of very small quantities of protein and has become a very popular
analytical technique in proteomics, as discussed below.
The procedures above describe treatment of an individual protein or mix-
tures of protein subunits obtained by breaking down quaternary structures of
individual proteins. More recently, researchers have been interested in numbers