Abstract
Introduction: Protein hydrolysis is the process by which peptide bonds are broken under acidic conditions at high temperatures, generating free amino acid residues. The resulting hydrolysates can then be analyzed by amino acid analysis to determine the amino acid composition and concentration of proteins. The hydrolysis procedure is the rate-limiting step for amino acid analysis. Microwave-assisted protein hydrolysis offers the potential to reduce hydrolysis time significantly.
Previous microwave studies indicate that high-temperature/short-time exposure can be applied to the hydrolysis of proteins for amino acid analysis. Currently for high-throughput protein hydrolysis, samples undergo vapor-phase hydrolysis in 6 N hydrochloric acid with phenol for either approximately 24 hours at 110°C or for 1 h at 150°C. Our studies evaluated various protein hydrolysis methods by comparing microwave irradiation to traditional hydrolysis conditions.
Materials and Methods: For the microwave-assisted hydrolysis studies, protein standards were hydrolyzed by microwave irradiation for 10 minutes at 175°C using the CEM Discover System (CEM Corp., Matthews NC). Protein standards were also hydrolyzed at either 110°C for 24 h or 150°C for 1 h in a Water Pico·Tag Workstation (Waters, Corp, Milford, MA). The hydrolysates were then analyzed using the Hitachi L-8800 Amino Acid Analyzer (Hitachi High Technologies America, San Jose, CA) .
Results: Protein standards hydrolyzed by microwave irradiation had comparable chromatographic profiles and amino acid recoveries to proteins that underwent traditional hydrolysis at 110°C for approximately 24 h or at 150°C for 1 h.
Conclusions: Protein hydrolysis by microwave radiation offered a significant time savings (10 minutes compared to up to 24 h), allowing for faster sample analysis. Microwave irradiation has been demonstrated as an acceptable alternative to the traditional protein hydrolysis conditions.