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Thermal Denaturation Analysis of Bovine Serum Albumin by Microfluidic Modulation Spectroscopy

November 6, 2020

Libo Wang, Jeff Zonderman, and Eugene Ma, RedShiftBio

The heat-induced secondary structure change of Bovine Serum Albumin (BSA)during thermal denaturation was investigated using Microfluidic Modulation Spectrometry (MMS), a novel technique that combines a microfluidic cell and a tunable mid-IR Quantum Cascade Laser source. BSA samples at 1 mg/mL, 20 mg/mL and100 mg/mL in water solution were incubated over a temperature range of 25 to 90 ºC. The differential absorbance spectra across the amide-I band for the samples at different concentrations and different incubation temperatures were then measured. For BSA samples at 20 mg/mL, at temperatures below 65 ºC, BSA maintained a native structure that consists of three major secondary structure components, specifically alpha helix at 1656 cm-1, beta turn at 1682 cm-1 and extended structure at1631 cm-1. At 65 ºC, BSA began to exhibit signs of denaturation with a slight decrease in alpha helix and the appearance of irreversible anti-parallel beta-sheet structures at 1618 cm-1 and 1692 cm-1.Above 65 ºC BSA lost more alpha helix structure and gained more beta-sheet structure, a trend that continued up to 80 ºC where BSA reached fully denaturation. For BSA at 1 mg/mL, a similar thermal denaturation trend was observed except the onset of denaturation appeared earlier, around 60 ºC. For BSA at 100 mg/mL, the onset of denaturation was observed at 65ºC. At this higher concentration, only samples incubated at temperatures 25through 70 ºC were tested due to the gelation behavior that occurred at higher temperatures. Our results show that Microfluidic Modulation Spectroscopy is a powerful technique for the measurement and analysis of protein secondary structure in samples over the concentration range between 1 mg/mL and 100 mg/mL.

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