Binding Ability of Bovine Milk Proteins to Mutagenic Heterocyclic Amine of 3-amino-1, 4-dimethyl-5H-pyrido[4, 3-b]indole

牛乳中の各種蛋白質、即ち whole casein, αs-casein, β-casein, κ-casein, β-lactoglobulin A, β-lactoglobulin B, α-lactalbumin, lactoferrin, lactoperoxidase を分離精製し、これらの蛋白質が発癌性ヘテロサイクリックアミンである 3-amino-1, 4-dimethyl-5Hpyrido[4, 3-b]indole (Trp-P-1) と給合するか否かについて実験を行った。さらに我々自身で分離調製した蛋白質と比較検討する為にSigma社製のα-casein, β-casein, κ-casein, β-lactoglobulin A, β-lactoglobulin B, α-lactalbumin, lactoferrin, lactoperoxidase ついても同様の実験を行った。凍結乾燥した蛋白質2mg, Trp-P1 20μgを0.4mlのpH7.4, 50mM燐酸バッファーに溶解し、37℃で10分間振盪し反応させる。つづいて、日本ミリポアリミテッド製のUFC3LGC00を用いて反応液中の非結合型 Trp-P-1 を限外濾過により分離し、この Trp-P-1 を HPLC 法により測定する。HPLC は日立 L-6000ポンプ・L-4000UV 検出機に GL-Science 製 Inertsil ODS-2(4.6×150mm)カラムを使用し、acetonitrile-H2O-triethylamine 50:50:0.05 で展開した。254nmに於ける吸収を SIC System Instruments 製Labchart 12で記録する。Whole casein:54.04%, αs-casein:40.02%, β-casein:56.24%, κ-casein:33.75% で反応液中の Trp-P-1 を比較的に高い割合で結合した。Sigma 社製の α-Casein:59.46%, β-casein:40.43%, κ-casein:23.75% で類似の傾向を示した。β-Lactoglobulin A:23.61%, β-lactoglobulin B:17.87%, β-lactoglobulin A・Sigma:15.78%, β-lactoglebulin B・sigma:3.65% であった。Lactoperoxidase:15.20%, lactoperoxidase・Sigma:6.41%, lactoferrin-a:22.53%, lacteferrin-b:6.92% であったが、α-lactalbumain, α-lactalbumin・Sigma, lacteferrin・Sigma は Trp-P-1 と結合しなかった。

The binding ability of bovine milk proteins with mutagenic heterocyclic amine was investigated. Binding was determined with 2 mg of protein and 20 g of 3-amino-1, 4-dimethyl-5H-pyrido[4, 3-b]indole (Trp-P-1) in 0.4 ml of pH 7.4, 50 mM phosphate buffer, at 37°C, in a shaker for 10 min. The unbound Trp-P-1 in protein-free ultrafiltrate was prepared using Ultrafree UFC3LGC00 and was analyzed by HPLC method. The binding of whole casein, αs-casein, β-casein and κ-casein were 54,03, 40.02, 56.24 and 33.75%, respectively. β-Lactoglobulin A, β-lactoglobulin B, lactoperoxidase, lactoferrin-a and lactoferrin-b were 23.61, 17.87, 15.20, 22.53 and 6.92% respectively. However, α-lactalbumin could not bind Trp-P-1 in this experiment. The binding of these milk proteins which were prepared by ourselves were compared with purchased proteins. They showed similar tendency to our proteins.