PITFALLS IN CHARACTERIZATION OF PROTEIN INTERACTIONS USING RADIOIODINATED CROSS-LINKING REAGENTS - PREPARATION AND TESTING OF A NOVEL PHOTOCHEMICAL I-125 LABEL TRANSFER REAGENT

Much attention has been focused on the study of protein interactions with radioiodinated photo-crosslinking reagents, and pitfalls in using this methodology are discussed. A new photochemical and cleavable heterobifunctional crosslinking reagent, succinimidyl N-14-(2-hydroxybenzoyl)-N-11-(4-azidobenzoyl)-9-oxo-8,11,14-triaza-4,5-dithiatetradecanoate (SHAD) was prepared, and its potential as a label transfer reagent was tested in model systems. SHAD was radioiodinated, and the labeled reagent (I-125-SHAD) was converted to an amide (I-125-HADM, as a mimicry of conjugation to protein 1) and photolyzed. When compared to the widely used SASD reagent (sulfosuccinimidyl 2-[[(4-azidosalicyl)-amino]ethyl]-1,3-dithiopropionate, Pierce), SHAD has a number of decisive advantages. The amide of I-125-SASD (I-125-ASDM) was generated and photolyzed, and it was found that at least 50% of the radioactivity is released from I-125-ASDM after 3 min of irradiation, whereas only approximately 10% is liberated from I-125-HADM under similar conditions. Furthermore, I-125-HADM was photolyzed in the presence of excess amine (mimicry of crosslinking to protein 2), and the product was cleaved by reduction (mimicry of label transfer). The transformations in the course of photolysis were monitored by UV spectroscopy and TLC analysis, and a high degree of reagent cleavage upon reduction was demonstrated. I-125-SHAD was used to crosslink Lys78-plasminogen and fibrin. I-125-SHAD was conjugated to Lys-plasminogen in the dark. Fibrinogen and thrombin were added, and Lys78-plasminogen was crosslinked to the fibrin clot by exposure to light. Irradiation for 5 min caused very little labeling of fibrin not crosslinked to Lys78-plasminogen; the total recovery of radioactivity was high, and the efficiency of the crosslinking was 30%. Under reducing conditions, it was found that all radioactivity was depleted from the Lys78-plasminogen-I-125-HAD conjugates in the dark, and label transfer showed that the labeling of the alpha-chain was significantly higher than that of the beta- and gamma-chains. Analogous experiments with I-125-SASD revealed that this reagent is much less suitable for photocrosslinking and label transfer studies than I-125-SHAD. The reactions were followed by polyacrylamide gel electrophoresis.