Simultaneous depletion of immunoglobulin G and albumin from human plasma using novel monolithic cryogel columns.

In this study, we aimed to develop an alternative matrix able to deplete the albumin (Alb) and immunoglobulin G (IgG) from blood plasma simultaneously to prepare plasma samples for large-scale applications of blood-related proteomics. As a first step, nano-protein A nanoparticles (nanoProA) were prepared and characterized. Subsequently, cibacron blue F3GA (CB) was immobilized onto the nanoProA's to enhance their specific affinity for Alb molecules. Finally, both nanoparticles, specifically, nanoProA and CB-nanoProA, were separately embedded into cryogel structures to combine advantages of the nanoparticles with those of the cryogels. The protein adsorption was optimized using aqueous Alb and IgG solutions separately. Subsequently, competitive protein adsorption was performed using a protein mixture prepared with Alb and IgG adhering to their plasma protein ratios. Because of the CB-immobilization, the Alb depletion performance of the cryogels increased whereas the IgG depleting performance decreased. Using the nanoProA, embedded cryogel removed 99.3% of the IgG, while using the CB-nanoProA embedded cryogel removed 97.5% of the Alb content. The simultaneous depletion performances of the cryogels for Alb and IgG were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, the monolithic cryogel-based adsorbents were classified as an alternative matrix to prepare plasma samples for proteomics applications at the preparative scale.

Polymeric amylase nanoparticles as a new semi-synthetic enzyme system for hydrolysis of starch.

α-Amylase (EC 3.2.1.1; α-D-1,4,glucan glucanohydrolase) catalyzes the hydrolysis of α-D-(1,4)-glucosidic linkages in starch, glycogen, and various malto-oligosaccharides, by releasing α-anomeric products. In this study, a novel method has been developed to prepare nanoprotein particles that carry α-amylase as a monomer by using a photosensitive microemulsion polymerization process. The nanostructured α-amylase with photosensitive features have been characterized by fluorescence spectroscopy, transmission electron microscopy (TEM) and Zeta Sizer. The fluorescence intensity of amylase nanoparticles was determined to be 658 a.u. at 610 nm and the average particle size of nanoamylase was found to be about 71.8 nm. Both free α-amylase and nanoparticles were used in the hydrolysis of starch under varying reaction conditions such as pH and temperature that affect enzyme activity and the results were compared to each other. Km values were 0.26 and 0.87 mM and Vmax values were 0.36 IU mg(-1) and 22.32 IU mg(-1) for nanoenzyme and free enzyme, respectively. Then, thermal stability, storage stability and reusability were investigated and according to the results, activity was preserved 60% at 60 °C; 20% at 70-80 °C temperature values and 80% after 105 days storage. Finally after 10 cycles, the activity was preserved 90% and this novel enzymatic polymeric amylase nanoparticle has showed considerable potential as reusable catalyst.