Trypsin Esterase Binding and Its Relation to the Radiation-Leukemia Protection (RLP) Factor 1

Abstract

The RLP (radiation-leukemia protection) activity of sheep spleen extracts is sharply eluted from the first Sephadex G-200 peak with the proteins of higher molecular weight (“19S peak”), whereas in serum the activity is more diffuse and considerable amounts appear in the lighter fractions (1). Berenblum and his co-workers have suggested that RLP is an α2-macroglobulin (α₂M) (2). As α₂M is readily detectable by its ability to bind trypsin causing protection of the esterase activity of the enzyme from inactivation by trypsin inhibitor (3), we have determined the distribution of the trypsin-esterase binding activity (TEBA) of serum and spleen extracts. The activity follows the RLP elution pattern for spleen but not for serum. Furthermore, the TEBA of serum and spleen exhibit differences in stability to heat and pH changes. In addition to the TEBA of serum and spleen, another characteristic property of α₂-macroglobulin, the trypsin inhibiting capacity (TIC) has also been investigated.

Materials and Methods. The preparation of sheep spleen extracts (40% homogenates in phosphate-buffered saline, pH 7.4, centrifuged 1 hr at 25,000 rpm in a Spinco rotor No. 30 in the cold) and fractionation of extracts and serum have been described (1). Fractionations were performed on a column of Sephadex G-200, equilibrated with phosphate-buffered saline. Both TEBA and TIC were determined by a modification of the methods of Ganrot (3, 4). The determinations were performed on 5-μl samples and the change in absorbancy at 410 mμ was recorded for at least 10 min in a Gilford spectrophotometer at 25°. Immunoelectrophoresis was performed in a Gelman apparatus in 1% agar at pH 8.6 in 0.1 M buffer.

Results. The fractionation on Sephadex of the substances precipitable upon 40% saturation of spleen extract with ammonium sulfate differs from that of the comparable serum preparation.