STRUCTURAL-CHANGES ASSOCIATED WITH THE SPONTANEOUS INACTIVATION OF THE SERINE PROTEINASE HUMAN TRYPTASE

Human skin tryptase, a serine proteinase stored within mast cell secre tory granules, rapidly loses enzymatic activity in solutions of physio logical salt concentration, pH, and temperature. The inactivation of t ryptase can be slowed and even reversed by addition of heparin, a high ly sulfated glycosaminoglycan also found in the secretory granules. Th ese properties may be relevant to tryptase regulation after secretion from mast cells. To further characterize the molecular changes underly ing the functional instability of tryptase, circular dichroism (CD) an d analytical ultracentrifugation were used to investigate structural c hanges during spontaneous inactivation. The CD spectra of active and s pontaneously inactivated tryptase are different, particularly in the r egion around 230 nm where active tryptase displays a distinct negative peak. This peak is also observed in the CD spectrum of bovine chymotr ypsin but not in trypsin, elastase, or chymotrypsinogen. Loss of activ ity resulting from spontaneous inactivation was accompanied by a dimin ution of the 230-nm signal. The kinetics for the signal loss appeared to be first-order and closely paralleled the rate of enzymatic activit y loss. Dextran sulfate, a highly sulfated polysaccharide, was capable of reactivating tryptase and restoring the CD signal. After 2 h of de cay (> 90% loss of activity), addition of dextran sulfate resulted in an almost immediate return of the CD signal to that of active tryptase . The return of the CD signal appeared to be more rapid than the retur n of enzymatic activity, thereby suggesting the presence of an unident ified step which is rate-limiting for activity return (and loss) and s ubsequent (prior) to the CD change accompanying activity loss. Ultrace ntrifugation analysis of tryptase showed a marked change in its associ ation state upon inactivation. Sedimentation equilibrium under stabili zing conditions demonstrated the presence of a single species with the molecular weight of a tetramer. After spontaneous inactivation, a mix ture of species was evident, which was characterized as monomers and t etramers in equilibrium. These results demonstrate that spontaneous in activation of tryptase is associated with reversible conformational ch anges and that a consequence of inactivation is the formation of a des tabilized tetrameric form. Although the molecular mechanism initiating these changes remains unclear, possible insights into the process are discussed on the basis of the similarity between the CD spectra of tr yptase and chymotrypsin.