Molecular Weight Determination of a Protease Extracted from <i>Mucor pusillus</i>: Comparison Methods

Abstract

Mucor pepsin, a protease used in milk coagulation, is purified by ion-exchange and by molecular exclusion on Sephadex G100. The molecular weight (MW) is determined by polyacrylamide gel electrophoresis under denaturing conditions in presence of sodium dodecyl sulfate (SDS) and by molecular exclusion chromatography. Approximate evaluation of molecular mass was conducted by elution of known MW proteins (BSA: 67 kDa, pepsin: 35 kDa and trypsin: 23.8 kDa) on Sephadex G-100 under the same conditions as the experimental sample. The electrophoretic profile shows that the active fraction studied appears as a single homogeneous band (monomeric form). According to the curve calibration, the molecular mass of the coagulant fraction is about 48 kDa. For Mucor, the observed MW value seems to be enigmatic. However, this result is confirmed by a proteomic analysis with close MW values obtained using conventional techniques. The protease studied by the Scafold ver. Software 2.0 and the analysis of the protein similarities indicate a MW of 46 kDa and the protease sequence of 427 amino acids.

Hybrid (Bovine Serum Albumin)/Poly(N-vinyl-2-pyrrolidone-co-acrylic acid)-Shelled Microbubbles as Advanced Ultrasound Contrast Agents

“…Mass spectrometry (MALDI-TOF) measurements of BSA@P­(VP-AA) in a broad m / z region and the linear recording mode (Figure a) made it possible to detect signals characteristic of the original BSA containing impurities of pepsin, trypsin, and protease. − In the region of low molecular weights, when registering the MALDI-TOF complex of BSA and P­(VP-AA) in the reflection mode, we observed a series of signals with an m / z difference close to 111, which is almost identical to that observed for P­(VP-AA) copolymer (Figure b). The latter circumstance indicates the destruction of the BSA@P­(VP-AA) complex under conditions of laser desorption from the DHB matrix.…”