A new algorithm for computation of initial velocity of enzyme reaction at the time zero is proposed. This algorithm makes it possible to reduce systematic error of measurements to the minimum, to estimate reaction velocity in testing samples regardless of the enzyme activity levels and to minimize assay time. The study is illustrated by an example of salivary alpha-amylase and standard reagent kit. The algorithm should not be applied if conjugated enzyme systems are used because there is a long initial lag-phase in the kinetic curve.
Introduction. The main consumers of commercial reagent kits for the study of metabolites and enzymes are clinical laboratories. Therefore, protocols for their using and calculating indices were developed mainly for blood and urine analysis. However, it is regularly necessary to determine metabolites concentration and enzymes activity to solve other tasks. Purpose of the work – as a case study estimation of glucose concentration and amylase activity in hemolymph plasma of the gastropod mollusc Achatina fulica to demonstrate how protocols for using some clinical reagent kits for determining metabolites concentration and enzymes activity can be optimized and how the scope of their application can be expanded. Material and Methods. The hemolymph plasma of the gastropod mollusc A. fulica served as the object of the study. Glucose concentration and amylase activity were determined in hemolymph plasma using clinical biochemical reagent kits from Spinreact (Spain). The standard and optimized protocols for determining glucose concentration are described. An optimized protocol for determining amylase activity has been described previously. Results. A. fulica is often used as a convenient model in experimental and practical developments of different areas of focus. The knowledge of diagnostically significant biochemical indices of hemolymph plasma of mollusks makes it possible to monitor the "state of health" of animals during their cultivation in laboratories and practical using. In contrast to the original protocol for determining glucose concentration, full calibration experiments were performed, with sample analysis time reduced to one minute. The optimized protocols for using reagent kits proposed in the article were tested to determine glucose concentration and amylase activity in the hemolymph plasma of A. fulica (n=16). The values were 0,42 ± 0,07 mM and 27,8 ± 0,6 IU/L, respectively. Conclusion. As a case study estimation of glucose concentration and amylase activity in hemolymph plasma of the gastropod mollusc Achatina fulica the effectiveness of the proposed ways of optimization of protocols for using some clinical reagent kits was demonstrated. The optimized protocols are suitable for determining the concentration of such metabolites as glucose and lactate as well as for analyzing the kinetics of various enzymes with direct measurements of their activity. The performed optimization allowed to expand the scope of application of biochemical reagent kits used in medicine, as well as to significantly reduce the time of analysis.
Two proteins, hemocyanin from the mollusk A. fulica and BSA, were used to show how the fourth derivatives of UV absorption spectra make it possible to reveal individual proteins characteristics associated with the presence of aromatic amino acids in their composition. To implement the method, the script was developed in the R programming language. This approach can be helpful in solving a variety of problems.
