Tryptic digest of three proteins of different size (bovine serum albumin, cytochrome c and ß-casein) was observed at 37°C and 50°C using both microwave and conventional heating (oil bath). The impact of the heating method on protein degradation and peptide fragment generation was analyzed using SDS-PAGE and MALDI-TOF. Samples were taken after 0 min, 1 min, 3 min, 5 min, 10 min, 30 min, 2 h and 16 h. The activity of trypsin was measured at different temperatures (37°C, 55°C, 60°C, 70°C and 80°C) after 1 min, 5 min and 10 min. Additionally, the effect of an increasing electric field strength on the protein structure of bovine serum albumin and trypsin was computational simulated.
|Setup||microwave reactor equipped with a fiber-optic thermometer; experiments performed using G4 reaction vessels with a volume of 0.5 - 2 ml and 5 mm x 3 mm stir bars; cooling performed with compressed air; all experiments performed under temperature control, not with constant power|
The experiments demonstrated comparable efficacy for time- and temperature- dependent tryptic digestion of the three model proteins independent of the heating source. No unspecific cuts caused by microwave exposure were identified. Furthermore, molecular mechanics calculations of the electric field strength required for influencing the tertiary structure of trypsin and bovine serum albumin have indicated that the electric field applied in commercial laboratory microwave instruments (ca. 3*104 V/m) is 3-4 orders of magnitude to low to effect the structure of enzyme or protein structure.
No differences occurred between microwave assisted and conventional heating. The authors hypothesize that formerly reported advantages of microwave assisted digest are due to incorrect temperature management.