Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 191

O

O

+

etc

H 3 N +

N C S CH C N CH C N

H

H

R 1 R 2

Phenylisothiocyanate

Peptide

O

S O

etc

NH C NH CH C N CH C N

H

H

R 1 R 2

6 M HCl

S

C NH O

+

etc

H 3 N + CH C N

NH

H

C CH

R 2

O

R 1

Phenylthiohydantoin-
amino acid derivative

Shorter peptide

Figure 7.5 The Edman degradation method, by which the sequence of a peptide/polypeptide may be elu-
cidated. The peptide is incubated with phenylisothiocyanate, which reacts specifi cally with the N-terminal
amino acid of the peptide. Addition of 6 mol l 1 HCl results in liberation of a phenylthiohydantoin-amino acid
derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its
constituent amino acid identifi ed by comparison to phenylthiohydantoin derivatives of standard amino acid
solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino
terminus may be identifi ed. This procedure is repeated until the entire amino acid sequence of the peptide
has been established

7.6 Endotoxin and other pyrogenic contaminants

Pyrogens are substances that, when they enter the blood stream, infl uence hypothalamic regula-
tion of body temperature, usually resulting in fever. Medical control of pyrogen-induced fever
proves very diffi cult, and in severe cases results in patient death.
Pyrogens represent a diverse group of substances, including various chemicals, particulate mat-
ter and endotoxin (LPS), a molecule derived from the outer membrane of Gram-negative bacteria.
Such Gram-negative organisms harbour 3-4 million LPS molecules on their surface, represent-
ing in the region of 75 per cent of their outer membrane surface area. Gram-negative bacteria