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Covalent Binding of Human ₂-Macroglobulin to Deglycosylated Ricin A Chain and Its Immunotoxins¹

Department of Microbiology and the Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas, Texas 75235

In this report we demonstrate that human ₂-macroglobulin (2M) reacts with deglycosylated ricin A chain (dgA) and its immunotoxins to form high molecular weight complexes (molecular mass approximately 800 kDa). This interaction has a t_1/2 at 37°C of 5 h and reaches completion at 24 h. Complexes of 2M-dgA cannot be dissociated by guanidine, sodium dodecyl sulfate, or low pH, but can be partially dissociated by reducing agents, such as 2-mercaptoethanol in the presence of sodium dodecyl sulfate. This indicates that dgA or dgA-containing immunotoxins are bound to 2M by disulfide bonds. The dgA-binding site on 2M and the mechanism underlying its interaction with dgA are different from those described for proteases or methylamine. 2M complexes do not bind to Blue-Sepharose 4B or anti-A chain-Sepharose, suggesting that the sites on dgA which bind Cibacron Blue or polyclonal anti-A chain antibodies are sterically blocked or modified by interaction with 2M. The interaction of 2M with dgA or its immunotoxins results in a 2- to 3-fold decrease in the activity of the dgA in both cell-free assays and cytotoxic assays. However 12 h after injection into mice, only 11% of immunotoxin was bound to 2M because of the slow kinetics of the interaction versus the more rapid t_1/2 of the immunotoxin in the circulation.

¹ This work is supported by NIH Grants CA-28149 and CA-41081 and Grant I-947 from the Welch Foundation.

² To whom requests for reprints should be addressed, at Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235.

Received 9/18/90. Accepted 12/20/90.

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