Structural property of soybean lunasin and development of a method to quantify lunasin in plasma using an optimized immunoassay protocol

Abstract

Lunasin is a 43-amino acid naturally occurring chemopreventive peptide with demonstrated anti-cancer and anti-inflammatory properties. The objectives of this study were to determine the effect of temperature on the secondary structure of lunasin, to develop a method of isolating lunasin from human plasma using an ion-exchange microspin column and to quantify the amount of lunasin using an optimized enzyme-linked immunosorbent assay. Lunasin was purified using a combination of ion-exchange chromatography, ultrafiltration and gel filtration chromatography. Circular dichroism showed that increased in temperature from 25 to 100 °C resulted in changes on the secondary structure of lunasin and its capability to interact with rabbit polyclonal antibody. Enzyme linked immunosorbent assay showed that lunasin rabbit polyclonal antibody has a titer of 250 and a specific activity of 0.05 mL/μg. A linear response was detected between 16 to 48 ng lunasin per mL (y=0.03x-0.38, R(2)=0.96). The use of diethylaminoethyl microspin column to isolate spiked lunasin in human plasma showed that most lunasin (37.8-46.5%) bound to the column eluted with Tris-HCl buffer, pH 7.5 with a yield up to 76.6%. In conclusion, lunasin can be isolated from human plasma by a simple DEAE microspin column technique and can be quantified using a validated and optimized immunoassay procedure. This method can be used directly to quantify lunasin from plasma in different human and animal studies aiming to determine its bioavailability.