High yield photoreagents for protein crosslinking and affinity labeling.

4-Nitrophenyl ethers are proposed as new high-yield photoreagents for protein crosslinking and affinity labeling. These are totally unreactive in the dark under biological conditions, but react quantitatively with amines at pH 8 upon irradiation with 366-nm light. The reaction of monoalkoxy-p-nitrobenzenes with an amine yields the corresponding free alcohol and substituted nitrophenylamine. In essence, the nitrophenyl group is transferred from the alcohol to the amine. Bifunctional affinity reagents of this type could be especially useful for placing the p-nitrophenyl chromophore adjacent to a binding site without blocking it. The corresponding 2-methoxy-4-nitrophenyl ethers react with amines by displacement of the methoxyl group. Thus, bifunctional reagents of this class could be photocrosslinkers. A maleimide-containing 2-methoxy-4-nitrophenyl ether was attached to human fetal hemoglobin at gamma-cysteine F9 stoichiometrically. Subsequent ultraviolet irradiation yielded a gamma-gamma crosslinked hemoglobin in 80% yield. The oxygenation properties of the derivative indicate that it is locked in a high affinity conformation and that all cooperativity is lost.