Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industry. Due to their versatile nature and ability to accept a broad range of substrates they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low sensitivity measurement of pH variations or visible changes on material properties, like hydration, and often require high amount of proteins. Fluorescent readouts on the other hand offer high contrast and even single molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report function of diverse lipases and phospholipases both in a microplate setup and at the single particle level on individual nanoscale liposomes using Total Internal Reflection Fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of enzymatic reaction is charged..
Short-chain lipid-linked fluorescent pH sensors allow for selective labelling of living cells and liposomes.
Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industry. Due to their versatile nature and ability to accept a broad range of substrates they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low sensitivity measurement of pH variations or visible changes on material properties, like hydration, and often require high amount of proteins. Fluorescent readouts on the other hand offer high contrast and even single molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report function of diverse lipases and phospholipases both in a microplate setup and at the single particle level on individual nanoscale liposomes using Total Internal Reflection Fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of enzymatic reaction is charged.
Lipid-conjugated pH sensors based on fluorophores coupled to lipids are a powerful tool for monitoring pH gradients in biological microcompartments and reconstituted membrane systems. This protocol describes the synthesis of pH sensors based on amine-reactive pHrodo esters and the amino phospholipid phosphatidylethanolamine. The major features of this sensor include efficient partitioning into membranes and strong fluorescence under acidic conditions. The protocol described here can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines. Graphical overview Synthesis of lipid-conjugated pH sensors based on amine-reactive fluorophore esters and the aminophospholipid phosphoethanolamine (PE)
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