125I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage.

Abstract

A radioactive crosslinking reagent, N-[4-(Pazido-m-[1251]iodophenylazo)benzoyl]-3-aminopropyl-N'-oxysulfosuccinimide ester, has been synthesized. The reagent is photoactivatable, water-soluble, cleavable through an azo linkage, and labeled with 125I at the carrier-free specific activity of 2000 Ci/mtnol. Any protein derivatized with the reagent is thus converted into an '251-labeled photoaffinity probe. Crosslinks are formed following photolysis with 366-nm light, and cleavage by sodium dithionite results in the donation of radioactivity to the distal partner in crosslinked complexes. The newly labeled proteins are then analyzed by gel electrophoresis and autoradiography. The compound was prepared by iodination of N-[4-(p-aminophenylazo)benzoylJ-3-aminopropionic acid using carrier-free Na'25I and chloramine-T, followed by azide formation and conversion to the water-soluble sulfosuccinimide ester. As a model system, protein A-Sepharose was derivatized with the reagent under subdued light. Each derivatized protein A molecule contained only one crosslinker. The derivatized protein A-Sepharose was then photolyzed in the presence of human serum and subsequently treated with sodium dithionite. Analysis of the serum by gel electrophoresis revealed that 1.1% of the radioactive label originally present on the protein A-Sepharose was transferred to the heavy chain of IgG, which was the most intensely labeled protein in the gel. The next most intensely labeled protein was IgG light chain, which incorporated radioactivity that was lowver by a factor of 3.6 than that of the heavy chain. These results demonstrated the specificity of the derivatized protein A-Sepharose as a photoaffinity probe. Photolabeling of IgG was the result of nitrene-mediated reactions and was not due to the incorporation of free 1251.Photoactivatable crosslinking reagents have been successfully used to detect plasma membrane receptors for insulin (1), epidermal growth factor (2), human choriogonadotropin (3), and the N-formyl chemotactic peptide (4) and to identify the nearest neighbors of many proteins, including fibronectin (5), calmodulin (6), fibrinogen (7), glycopeptides (8), and ribosomal proteins (9) (for other previous studies, see refs. 10-12). In some cases (1-4, 6, 8) relatively small proteins and peptides were labeled with 125I, derivatized with a photoactivatable reagent, and crosslinked to their putative receptors. However, crosslinks were not cleaved and high molecular weight complexes were assumed to contain the 125I-labeled peptide and its receptor. The above approach is more difficult when used to detect receptors for larger proteins, due to the inability of crosslinked complexes to enter polyacrylamide gels. Cleavable, photoactivatable crosslinking reagents were therefore developed (10-12), but most of the reagents are cleavable through a disulfide bond and are therefore subject to mercaptan-disulfide interchange with protein-SH groups (13,14). In addition, they cannot be used in the presence of thiol reducing agents. We the...

Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking.

“…Mouse oocyte ZP3 (2 ug) was purified as described (8,17) and treated with 500 ,uCi (1 ,uCi = 37 kBq) of '25I-labeled DennyJaffee reagent, N-{4-(p-azido-m-[1251]iodophenylazo)benzoyl}-3-aminopropyl-N'-oxysulfosuccinimide ester (New England Nuclear; 2000 Ci/mmol), in 50 ,1u of sodium bicarbonate (pH 8.3) for 1 hr at 25°C in the dark (14). The crosslinker reacts with protein amino groups during this step.…”

“…Mouse oocyte ZP3 (2 ug) was purified as described (8,17) and treated with 500 ,uCi ( (14). The crosslinker reacts with protein amino groups during this step.…”

“…The remainder was treated with sodium dithionite to cleave crosslinks between ZP3 and sperm proteins. At 15-min intervals, 1.5 M sodium dithionite was added to samples to a final concentration of 0.2 M as described (14). After the final addition of sodium dithionite, samples were incubated at 25°C for 10 hr.…”

“…Identification of the sperm-head protein(s) to which 1251-DJ-ZP3 was covalently bound following photoactivation was determined by SDS/PAGE and autoradiography after treatment of the samples with sodium dithionite. The latter treatment resulted in reduction of the 125I-DJ-ZP3 azo linkage (14,15) and, consequently, in transfer of radiolabel from derivatized ZP3 to sperm protein. Results of these experiments are shown in Fig.…”

“…Purified ZP3 was covalently modified with a radiolabeled, photoactivatable, heterobifunctional crosslinker ("Denny-Jaffee reagent") (13)(14)(15), incubated with acrosome-intact and acrosome-reacted sperm, and then subjected to photolysis. …”