Chemistry Reference
In-Depth Information
(
3
H)
HO
(CH
2
)
1-3
NH
N
3
OH
(
3
H)
O
2
N
36
(
3
H)
HO
37a and b
H
N
3
(
3
H)
O
HN
OH
38
O
2
N
O
O
NH
N
3
Br
O
*
O
SCHEME 3.18.
attached to the bromoacetate moiety, and then, following irradiation, attached to the
PAL moiety. This technique has been used to identify previously unrecognized
proteins that bind to vitamin D
3
.
109
A potential pitfall in designing PAL experiments
of this type is that ether- or amine-linking chains,
36
and
37
, are more suitable than
38
are ester- or amide-linking chains,
. The latter type of linking chains are
susceptible to hydrolysis either during the cross-linking experiment or during the
analysis that follows, and if these cross-linking chain are broken, it can become much
more difficult or impossible to identify the cross-linked proteins.
Intact proteins and polynucleotides can be labeled by alkylation with reactive
halides at nucleophilic sites to form useful PAL reagents. One example of this type of
strategy is the selective attachment of 4-azido-2-nitrophenyl amine PAL units to
radio-labeled (
125
I) synthetic analogs of bovine parathyroid hormone at lysine
residues along the peptide chain by treatment with FNPA as indicated in Scheme 3.19
(gray Lys units).
110
The conditions selected for this labeling minimized attachment to the
N
-terminal
amino group. On irradiation in the absence of competing hormone, this modified
parathyroid hormone became cross-linked to four-six components of the renal
receptor membrane, but in the presence of competing hormone, one of these
membrane components failed to become cross-linked indicating that this was the
primary hormone binding site. This general strategy of using the natural substrates
modified by incorporation of PAL units, and determining the degree to which the
natural substrate competes with the PAL modified substrate is widely used to identify
binding sites of biomolecules.
In another variation of PAL-modified natural molecules, tRNA of some micro-
organisms contains extremely nucleophilic bases that can be easily attached to PAL
