This only partially explains the sensogram of pepsin in fig. 6A and does not explain why the signal went down below the baseline unless there was also the minor cleavage of HE- 4 which was not detectable in SDS-PAGE or WB because SPR would be more sensitive to even small amount of cleavage. The sensograms of other proteases were characteristic of a normal protein-protein interaction. HE-4 exists as a disulfide bonded trimer in human seminal fluid as inferred from SDS-PAGE in reducing and non-reducing conditions. This is not unheard of in proteins and many human and some viral proteins employ intermolecular disulfide linkages to form tertiary structure. HE-4 has eight predicted disulfide bonds per monomer of protein as it is a small protein, these bonds and intermolecular disulfide linkages would be expected to give it a compact structure resistant to denaturing agents. Accordingly, it found that HE-4 is resistant to pH, heat and even SDS in protease inhibition assay taking trypsin as a model protease. Seminal fluid has a high concentration of zinc and it regulates the function of several seminal fluid proteins like PSA and sememnogelin.Therefore, we measured Rh value and AM 92016 hydrochloride activity of HE-4 in the presence of zinc and we observed lower Rh than that of purified native protein in Tris buffer. Introduction of EDTA in twice the molar ratio of zinc increased the Rh value a little bringing it closer to native HE-4. Trypsin inhibition activity of HE-4 reduced slightly in the presence of zinc while the addition of EDTA to the Zn supplemented HE-4 recovered the activity a little as shown in figure 4D. The activity lost by 2 mM ZnCl2 could be completely rescued by addition of increasing concentrations of EDTA. At this stage evidence for mechanism of effect of zinc on activity and structure of HE-4 is inconclusive. However, this idea is being pursued further in our laboratory. Disulfide bonds play an important role in BW 723C86 hydrochloride maintaining the native conformation of a protein, which in turn provide stability/ resistance towards pH and temperature treatments. HE-4 contains 8 disulfide bonds. Therefore, it was of interest to evaluate the effect of DTT reduction on the trypsin inhibitory activity of HE-4. Even at.05 mM there was a significant reduction in trypsin inhibitory activity of HE-4 and at 1 mM HE-4 lost all its activity against trypsin. Disulfide bond reduced HE-4 was oxidized and refolded but HE-4 was unable to restore the activity and at 16 hr. time-point only 3% activity was restored. PNGase F treatment of HE-4 produced a shift of approximately 2.5 kDa which is considerable given the molecular weight of the monomer. This also partly explains the observed heat and pH resistance of the protein. Asn44 has been reported to be glcyosylated in salivary HE-4 previously, and no other glycosylation site has been either predicted or reported.