Selective and asymmetric action of trypsin on the dimeric forms of seminal RNase

• Published in: Protein science Vol. 7; no. 12; pp. 2653 - 2658
• Main Authors: Lorenzo, Claudia De, Piccoli, Renata, Maro, Antimo Di, Pucci, Piero, D'alessio, Giuseppe, Piaz, Fabrizio Dal
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.12.1998
• Subjects: Animals; BS‐RNase; Cattle; Dimerization; domain swapping; Hydrolysis; limited proteolysis; Male; mass spectrometry; Mass Spectrometry - methods; Models, Molecular; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Conformation; Ribonucleases - chemistry; Ribonucleases - isolation & purification; Ribonucleases - metabolism; Semen - enzymology; trypsin; Trypsin - chemistry; Trypsin - metabolism
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560071219

Dimeric seminal RNase (BS‐RNase) is an equilibrium mixture of conformationally different quaternary structures, one characterized by the interchange between subunits of their N‐terminal ends (the MXM form); the other with no interchange (the M=M form). Controlled tryptic digestion of each isolated quaternary form generates, as limit digest products, folded and enzymatically active molecules, very resistant to further tryptic degradation. Electrospray mass spectrometric analyses and N‐terminal sequence determinations indicate that trypsin can discriminate between the conformationally different quaternary structures of seminal RNase, and exerts a differential and asymmetric action on the two dimeric forms, depending on the original quaternary conformation of each form. The two digestion products from the MXM and the M=M dimeric forms have different structures, which are reminiscent of the original quaternary conformation of the dimers: one with interchange, the other with no interchange, of the N‐terminal ends. The surprising resistance of these tryptic products to further tryptic action is explained by the persistence in each digestion product of the original intersubunit interface.