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Novel method for measuring aquatic bacterial productivity using D10-leucine based on protein synthesis rate

Kenji Tsuchiya*, Tomoharu Sano, Noriko Tomioka, Komatsu Kazuhiro, Akio Imai, Kazuhide Hayakawa, Takamaru Nagata, Takahiro Okamoto, Victor S. Kuwahara, Ayato Kohzu

*Corresponding author:

ABSTRACT: For measuring bacterial production, the tritium-labeled leucine (3H-Leu) method is the most widely used. Although the method provides methodological simplicity and high sensitivity, the employment of radioactive isotopes is often restricted by regulation, particularly in field settings. In this study, we developed a non-radioactive method for measuring bacterial productivity based on protein synthesis rate, using deuterium-labeled leucine ((CD3)2CDCD2CD(NH2)COOH; D10-Leu), where the proposed method was compared and verified with 3H-Leu method. The procedures of the proposed method were, 1) incorporation of D10-Leu by bacteria, 2) acid hydrolysis (HCl) to amino acids and 3) quantification of D10-Leu (m/z 142.10) by liquid chromatography mass spectrometry (LC-MS/MS). In the LC-MS/MS analysis, we detected a larger amount of D9-Leu (m/z 141.10) and D8-Leu (m/z 140.10) than that of D10-Leu, suggesting that incorporated D10-Leu was rapidly metabolized such as in deamination and aminotransferase reactions. The incorporation rates of D10-Leu, D10-Leu + D9-Leu (D10+D9-Leu), D10-Leu + D9-Leu + D8-Leu (D10+D9+D8-Leu) were significantly positively correlated to that of 3H-Leu, confirming the validity of the proposed method. Since D7-Leu (m/z 139.10) could not be detected, the amount of exogenous leucine incorporated into protein can be estimated through D10+D9+D8-Leu measurement accurately. The new compound-based quantification method using stable isotope-labeled leucine can be a powerful tool to estimate pure protein synthesis rate for measuring bacterial production.