Selective cleavage of pepsin by molybdenum metallopeptidase.

In this study, the cleavage of protein by molybdenum cluster is reported for the first time. The protein target used is porcine pepsin. The data presented in this study show that pepsin is cleaved to at least three fragments with molecular weights of ∼23, ∼19 and ∼16 kDa when the mixture of the protein and ammonium heptamolybdate tetrahydrate ((NH(4))(6)Mo(7)O(24)·4H(2)O) was incubated at 37°C for 24h. No self cleavage of pepsin occurs at 37 °C, 24h indicating that the reaction is mediated by the metal ions. N-terminal sequencing of the peptide fragments indicated three cleavage sites of pepsin between Leu 112-Tyr 113, Leu 166-Leu 167 and Leu 178-Asn 179. The cleavage reaction occurs after incubation of the mixture of pepsin and (NH(4))(6)Mo(7)O(24)·4H(2)O) only for 2h. However, the specificity of the cleavage decreases when incubation time is longer than 48 h. The mechanism for cleavage of pepsin is expected to be hydrolytic chemistry of the amide bonds in the protein backbone.

Photocleavage of avidin by a new pyrenyl probe.

In this study, a new small-molecule-based reagent was designed to recognize and bind to specific site in protein. A new pyrenyl probe, d-biotinyl-1(1-pyrene)methylamide (Py-biotin) was designed and synthesized by coupling of d-biotin to 1(1-pyrene)methylamine hydrochloride. Binding studies and site-specific photocleavage of avidin by Py-biotin were demonstrated. Binding of Py-biotin to avidin was studied using absorbance and fluorescence spectroscopic techniques. Red shifts of the absorption peak positions of the pyrenyl chromophore followed by hyperchromism were observed upon binding to avidin. The photocleavage of avidin was achieved when a mixture of the protein, Py-biotin, and an electron acceptor, cobalt(III) hexammine trichloride (CoHA), was irradiated at 342nm. No reaction occurred in the absence of the probe, CoHA, or light. N-terminal sequencing of the peptide fragments indicated a cleavage site of avidin between Thr 77 and Val 78. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.