title: 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.
authors.affiliation: Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, USA.
authors.forename: Vermont P
authors.initials: VP
authors.lastname: Dia
authors.forename: Sarah
authors.initials: S
authors.lastname: Frankland-Searby
authors.forename: Francisco Laso
authors.initials: FL
authors.lastname: del Hierro
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Garcia
authors.forename: Elvira Gonzalez
authors.initials: EG
authors.lastname: de Mejia
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D017433:Protein Structure, Secondary; D011817:Rabbits; D030262:Soybean Proteins / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D013025:Glycine max / Q000737:chemistry*; D013696:Temperature
chemical_list: C120514:GM2S-1 protein, soybean; D030262:Soybean Proteins