i. Theory
To simulate the degradation by protease, we used Michaelis-Menten kinetics model to express the rate equation. It is useful to describe enzymatic reactions, especially the one without ligand participation, such as proteolysis with proteases whose mechanism is first to find out the specific active site and then react to break it down. [3]
The proteolysis process is irreversible in vivo because the product, linear form protein, is degraded quickly. We used differential equation to describe the major proteolysis process:
Where [P ]: the concentation of Pantide, Vm,p : the maximum reaction rate of proteolysis, which is equal to the product of the concentration of total enzyme and turnover number kcat of specific protease and substrate, KM,p : Michaelis constant, which is the substrate concentration at which the reaction rate is half of Vm,p
We had assumed that the Vm,p is a constant because the proteases in vivo are in active and in equilibrium, and Vm,p represents the equivalent value for all kinds of protease in environment.
ii. Experimental proof
We designed two experiments to test the enzymatic stability towards protease of Hv1a and Hv1a-lectin. One was to observe the degrade level of both native and linear types of proteins applied by protease for one day, and the other one was to obtain the curve of the degradation rate of only linear form proteins in the period of four hours, because of the resistance against the protease.
For the first experiment, we dissolved the protein solutions in neutral PBS (pH=7.5) solvent, applied with 0.25% trypsin-EDTA(1:250), a serine protease, and then incubated the samples in work temperature 37℃ for one day. (Figure 4, Figure 5)
Compared with the sample incubated for one day without trypsin treatment, native Hv1a and Hv1a-lectin showed the resistance to trypsin protease (111% and 100%), but linear proteins were degraded by proteolysis (67% and 18%).
We next tested the proteolysis rate of only linear proteins in the period of four hours, and we drew the Lineweaver–Burk plot (also called “double reciprocal plot”) (Figure 6) to obtain Vm,p and KM,p for linear form Hv1a and Hv1a-lectin. (Table 1)
KM,p , and y-intercept means the reciprocal of Vm,p .”>
Vm,p and KM,p of linear form Hv1a and Hv1a-lectin.”>