Changes Occurring on the Activity of Salivary Alpha-Amylase Proteoforms in Two Naturalistic Situations Using a Spectrophotometric Assay

SIMPLE SUMMARY: Salivary alpha-amylase (sAA) is considered a biomarker of acute stress since this enzyme is released in saliva after autonomic nervous system activation, in response to psychological or physical stress situations. This enzyme has different isoforms that could be differentially expressed depending on the stressful situation. The aims of the present research were (1) to develop and validate an easy and fast method to estimate the activity of the major sAA proteoforms (both non-glycosylated and glycosylated proteoforms) in saliva samples, and (2) to evaluate the possible changes occurring in the activity of both proteoforms when measured by this method in two different stress models (physical effort and psychological challenge). This new method was precise and, when applied to the different stress models, allowed to detect changes of different magnitudes in both proteoforms. Therefore, this research opens a new field for the evaluation of isoforms of sAA as potential biomarkers of stress. ABSTRACT: This study aimed to evaluate the changes in the activity of total salivary alpha-amylase (TsAA) and both the non-glycosylated and glycosylated salivary alpha-amylase proteoforms (NGsAA and GsAA, respectively) in physical and psychological stress models, estimated using a simple and easily set-up method. The method used was a spectrophotometric assay with 2-chloro-4-nitrophenyl-α-D-maltotriose (CNPG3) as a substrate, incubated with Concanavalin A (ConA) to remove most of the glycosylated protein from the sample. This method allowed the measurement of TsAA and estimation of NGsAA and GsAA activities with imprecision lower than 10%. When this method was applied to two different stress models, differences in the responses of the proteoforms were observed, with the NGsAA activity showing changes of higher magnitude after stress induction than the GsAA activity, and the highest correlation with the State–Trait Anxiety Inventory Scale in the Trier Social Stress Test (TSST). In conclusion, the activity of the two main sAA proteoforms can be easily estimated in saliva, and their measurement can provide additional information on TsAA activity in physical or psychological stress situations.