Purification And Characterization Of A Recombinant Trypsin Inhibitor In Buckwheat

Proteinase inhibitors are widely distributed in nature including many kinds of animals, plants and microorganisms. They play important roles in maintaining the interaction balance of enzyme proteolysis in vitro and in vivo. The biological role of protease inhibitors is suggested that they may perform three main functions—serving as storage proteins, being regulators of activity of endogenous proteinases and agents protecting plants against insects and pathogenic microbe. According to the different enzymes inhibited, the inhibitors are generally classified into four types—serine proteinase inhibitor, cysteine proteinase inhibitor, asparagine proteinase inhibitor and metalloproteinase inhibitor. Among these serine proteinase inhibitors are most extensively studied in the primary structure, the composition of amino acid, their interactions with serine proteinase and possible biological roles. Naturally-occurring proteinase inhibitors have been isolated from various plants and characterized in terms of their chemical and physicochemical properties by many investigators. Especially trypsin inhibitors from soybean and mammalian pancreas have been extensively studied for their physiological roles, inhibitory mechanisms and structures. Trypsin inhibitors, having the inhibitory activity against trypsin, belong to serine proteinase inhibitor. However, few reports exist on buckwheat trypsin inhibitors (BTI).Buckwheat (Fagopyrum esculentum Moench) is planted in many regions covering Asia, Europe, North America, Austria, South Africa, and other countries in the world, but its birthplace locates in the southwest region of China. In many regions it even is the major food. Nutrition abounds in buckwheat, whose seeds contain some nutritive substances nonexistent or insufficient in other cereal crops. Buckwheat is known as a valuable source of protein, and chemical analyses of hydrolysis indicate that the amino acid composition of buckwheat is nutritionally superior to that of cereal grains. Buckwheat flour contains 18 varieties of amino acid, among which 8varieties essential to human body are abundant. Buckwheat is an excellent medical plant as well as a nutrient-abundant crop. Its flour and leaf contain large quantity of flavonoid compounds. Increasing attention has been paid to Buckwheat. It is an important food for both humans and domestic animals, but its low digestibility, possible due to the presence of inhibitors as an antinutritional factor, has been reported. The antiproliferactive effect of tartary buckwheat trypsin inhibitor (TBTI) was determined by MTT assay on human acute myelogenous leukemia (AML) HL-60 cells. The data showed that TBTI obviously inhibited the proliferation of HL-60 leukemia cells, whereas less cytotoxicity on normal human peripheral blood mononuclear cells. The result suggests that TBTI may have potential for treating human leukemia as an interesting new antineoplastic agent. In scientific research field, discoveries of some particularly physiological and biochemical characteristics in buckwheat have aroused great interests in scientists to further study the mechanisms of the corresponding phenomena. As an important bio-active protein, BTI should be paid to more attention.In this paper, purification of the recombinant buckwheat trypsin inhibitor (rBTI) was obtained by a purification procedure that included affinity chromatography on Ni2+- NTA column and gel filtration chromatography on Superdex G-75 10/30 HR. The analysis of some properties showed that rBTI was similar to the native trypsin inhibitors in buckwheat seeds. It had a strong inhibition against trypsin and the mole ratio was about 1:1.3. The relative molecular weight of the inhibitor was determined using an ionization time-of-flight mass spectrometer. It had a relative molecular weight of 9 322.1 Da. Isoelectric-focusing (IEF) was applied to measure the electric point (pi) of rBTI, using Pharmalyte with a pH range of 3-10 and an IEF Calibration Kit (pH 3-10) as markers. The result showed that it had a pi of 6.2. This was consistent with the amino acid component that more acidic amino acid residues existed than basic amino acid residues analyzed by the gene sequence of rBTI. The stability of the purified protein at different pH values was assayed and it had a pH optimum of 8.0. Thermal stability of rBTIwas also studied. The purified inhibitor was found to be heat-stable when subjected to boiling (100 °C) in the pH range 3.0-8.0. After 120 min of heat treatment (100 °C) the inhibitor remained over 70% of its trypsin inhibitory activity. At alkaline pH, the purified inhibitor was heat labile, losing antitryptic activity rapidly upon heating to 100 °C. These data suggested that rBTI was heat-stable at neutral but it could be rapidly inactivated by heat under alkaline conditions. The high contents of acidic amino acids and valine residues, low contents of half-cystine residues and the lack of tyrosine and histidine residues suggested that rBTI belongs to the permanent inhibitors. Site directed mutagenesis was performed to obtain the mutant of Arg45-Asp46 for Ile45-Val46. The result that the mutant lost about 90% of its antitryptic activity suggested that the reactive site of the inhibitor may contain an Arg45-Asp46 bond. According to the amino acid compositions and the molecular weight rBTI was a member of the potato proteinase inhibitor I family.Considering on the whole, we obtained a small molecular recombinant trypsin inhibitor and investigated its biochemical and physiochemical properties. Lot of potentially beneficial qualities possessed by BTI has indicated its promising future in scientific research field and practical application in nutrition and medicine. Our work enriches the scientific research of rBTI and it will be useful for further studying in the fields of nutrition, medicine and plant self-protection and so on.