Effects of thiodiethanol on the kinetics of peptide hydrolysis by carboxypeptidase-Y

Optimization of a solvent system that can be used in Electrohydrodynamic Mass Spectrometry (EHMS) and effects of that solvent system on kinetics of an enzyme is the topic of present investigation. Thiodiethanol (TDE) was used as the low volatility organic solvent. Carboxypeptidase-Y was used because of its versatility to cleave most amino acids. Three carbobenzyloxy-protected dipeptides were hydrolysed, and the absorbance of the released amino acids was monitored with a diode array UV/VIS spectrophotometer. The kinetics parameters such as rate constant (k), Michaelis constant (K_m) , and maximum velocity (V_max) were determined at 0, 10%, 20%, and 40% TDE in phosphate buffer and 0.5 M sodium chloride. A pentapeptide, Leu-Enkephalin, was also hydrolysed at 0 and 20% TDE in phosphate buffer, under similar conditions, and the rate constants were estimated assuming first order kinetics, using a nonlinear approximation procedure. The results show that TDE inhibits enzyme hydrolysis. For a given concentration of enzyme, as the amount of TDE in the solvent system increased, rate constant for hydrolysis k, and V_max decreased, and K increased. These results prove that TDE can be used in EHMS, with a maximum of 20% TDE in buffer.