Enzymes adsorbed onto model surfaces

STORY ETAL. Enzymes Adsorbed onto Model Surfaces... 

Adsorption of the enzymes subtilisin BPN and lysozyme onto model hydrophilic and hydrophobic surfaces was examined using adsorption isotherm experiments, infrared reflection-absorption spectroscopy (IRRAS), and attenuated total reflectance (ATR) infrared (IR) spectroscopy. For both lysozyme and BPN, most of the enzyme adsorbed onto the model surface within ten seconds. Nearly an order-of-magnitude more BPN adsorbed on the hydrophobic Ge surface than the hydrophilic one, while lysozyme adsorbed somewhat more strongly to the hydrophilic Ge surface. No changes in secondary structure were noted for either enzyme. The appearance of carboxylate bands in some of the adsorbed BPN spectra suggests hydrolysis of amide bonds has occurred. 

A report by Liu and coworkers described layer-by-layer (LBL) coatings that have been assembled on the inner surfaces of the microchip [90]. Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. Trypsin was adsorbed in the multilayer membrane composed of CS/HA assembled multilayers. The resulting peptide analysis has been carried out by MALDI-TOF-MS. The maximum proteolytic velocity of the adsorbed trypsin was 600 mM/min/[ig, thousands of times faster than that in solution. BSA, MYO, and Cyt-c were used as model substrates for the tryptic digestion. The standard proteins were identifled at a low femtomole per analysis at a concentration of 0.5 ng/pl with the digestion time <5 s. This simple technique may offer a potential solution for low-level protein analysis. 

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