Non-specific normal immunosuppressive protein (Nip): purification and further biochemical characterization

The non-specific normal immunosuppressive protein (Nip) has been described in our laboratory and its biological activity was extensively studied. In the present study, further purification analyses of Nip were conducted. Fractionation of Nip by Ultrogel AcA 34 column resulted in peak (I) that displayed Nip activity in that it exhibited marked inhibition of in-vitro blastogenic responses of human lymphocytes to mitogenic stimulation, in vitro it inhibited EL4 tumour-cell proliferation. Partial dissociation of Ultrogel peak I on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) resulted chiefly in three bands: one of high molecular weight, which is considered to be the carrier, an intermediate band of 50,000-60,000 (under non-reducing conditions) and a band of high mobility and low molecular weight approximately 6,000-14,000. Further fractionation of peak I on Sepharose-6B in 6 M guanidine-HCl resulted in two main peaks. The biological activity resided in the second peak, which corresponded to the low molecular weight fraction of 6,000-14,000. Nip is suggested to be a complex molecule comprised of a low molecular weight peptide or glycopeptide, which displays the biological activity, and a macromolecular glycoprotein carrier, which conserves its stability.