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High yield photoreagents for protein crosslinking and affinity labeling
P C Jelenc1, C R Cantor, S R Simon
1Department of Chemistry, Columbia University, New York, New York 10027.
Summary
New 4-nitrophenyl ethers act as efficient photoreagents for protein crosslinking and affinity labeling. These compounds react with amines under UV light, enabling precise modification of proteins like hemoglobin.
Area of Science:
- Biochemistry
- Photochemistry
- Molecular Biology
Background:
- 4-Nitrophenyl ethers are explored as novel photoreagents.
- These reagents demonstrate high reactivity with amines upon UV irradiation.
- They are stable under dark biological conditions.
Purpose of the Study:
- To introduce 4-nitrophenyl ethers as high-yield photoreagents for protein crosslinking and affinity labeling.
- To investigate the mechanism of reaction with amines.
- To demonstrate the utility of bifunctional reagents for protein modification.
Main Methods:
- Synthesis and characterization of 4-nitrophenyl ethers.
- Photochemical reaction studies with amines at pH 8 and 366-nm light.
- Attachment of a maleimide-containing ether to human fetal hemoglobin.
- UV irradiation of modified hemoglobin to induce crosslinking.
Main Results:
- 4-Nitrophenyl ethers react quantitatively with amines, transferring the nitrophenyl group.
- Bifunctional reagents enable photocrosslinking, as demonstrated with hemoglobin.
- A gamma-gamma crosslinked hemoglobin derivative was formed in 80% yield.
- The crosslinked hemoglobin exhibited locked high-affinity oxygenation with lost cooperativity.
Conclusions:
- 4-Nitrophenyl ethers are effective and versatile photoreagents for protein modification.
- These reagents facilitate precise protein crosslinking and affinity labeling.
- Photocrosslinked hemoglobin shows altered oxygen-binding properties, indicating conformational changes.