Enzyme solid-state support assays: a surface plasmon resonance and mass spectrometry coupled study of immobilized insulin degrading enzyme.
Solid-support based assays offer several advantages that are not normally available in solution. Enzymes that are anchored on gold surfaces can interact with several different molecules, opening the way to high throughput array format based assays. In this scenario, surface plasmon resonance (SPR) and mass spectrometry (MS) investigations have often been applied to analyze the interaction between immobilized enzyme and its substrate molecules in a tag-free environment. Here, we propose a SPR-MS combined experimental approach aimed at studying insulin degrading enzyme (IDE) immobilized onto gold surfaces and its ability to interact with insulin. The latter is delivered by a microfluidic system to the IDE functionalized surface and the activity of the immobilized enzyme is verified by atmospheric pressure/matrix assisted laser desorption ionization (AP/MALDI) MS analysis. The SPR experiments allow the calculation of the kinetic constants involved for the interaction between immobilized IDE and insulin molecules and evidence of IDE conformational change upon insulin binding is also obtained.
Grasso
Giuseppe
G
Dipartimento Scienze Chimiche, Università di Catania, v.le A. Doria 6, 95125, Catania, Italy. grassog@unict.it
Bush
Ashley I
AI
D'Agata
Roberta
R
Rizzarelli
Enrico
E
Spoto
Giuseppe
G
D000818:Animals; D001274:Atmospheric Pressure; D002417:Cattle; D004800:Enzymes, Immobilized / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D007328:Insulin / Q000737:chemistry; D007339:Insulysin / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D044085:Microfluidics; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D020349:Surface Plasmon Resonance / Q000379:methods*; D013995:Time
D004800:Enzymes, Immobilized; D007328:Insulin; D006046:Gold; D007339:Insulysin