Purification and Characterization of the 7S Vicilin from Korean Pine (Pinus koraiensis) TENGCHUAN JIN, † SILVIA M. ALBILLOS, ‡ YU-WEI CHEN, † MAHENDRA H. KOTHARY, § TONG-JEN FU, | AND YU-ZHU ZHANG* ,† Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616; Illinois Institute of Technology, National Center for Food Safety and Technology, 6502 South Archer Road, Summit-Argo, Illinois 60501; Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, Maryland 20708; and U.S. Food and Drug Administration, National Center for Food Safety and Technology, 6502 South Archer Road, Summit-Argo, Illinois 60501 Pine nuts are economically important as a source of human food. They are also of medical importance because numerous pine nut allergy cases have been recently reported. However, little is known about the proteins in pine nuts. The purpose of this study was to purify and characterize pine nut storage proteins. Reported here is the first detailed purification protocol of the 7S vicilin-type globulin from Korean pine (Pinus koraiensis) by gel filtration, anion exchange, and hydrophobic interaction chromatography. Reducing SDS-PAGE analysis indicated that purified vicilin consists of four major bands, reminiscent of post-translational protease cleavage of storage proteins during protein body packing in other species. The N-terminal ends of vicilin peptides were sequenced by Edman degradation. Circular dichroism (CD) and differential scanning calorimetry (DSC) analyses revealed that pine nut vicilin is stable up to 80 °C and its folding-unfolding equilibrium monitored by intrinsic fluorescence can be interpreted in terms of a two-state model. KEYWORDS: Pinus Koraiensis; pine nut; 7S globulin; vicilin-like seed storage protein; food allergen INTRODUCTION Pines (Pinus genus) are gymnosperms. They are evolution- arily important as they represent an ancient evolutionary branch; they are economically important as they provide a source of timber, wood pulp, and food, and they are medically relevant as pine nut allergy poses a risk to a subpopulation of consumers. Gymnosperms are evolutionarily ancient, arising as early as 300 million years ago. The conifers, a division to which the pines belong, separated from flowering plants (angiosperms) about 100 million years ago and by most measures have evolved very slowly and conservatively (1–3). Pines therefore represent an ancestral branch in the tree of life. Pines are among the most widely distributed and prominent genera of trees in the world. They extend throughout the Northern Hemisphere from the equator to the Arctic Circle and are the dominant vegetation over large areas (4). Pines are the most important forest trees and crop species in the United States and around the globe. One single species, Loblolly pine (Pinus taeda L.), alone provides ∼16% of the world’s annual timber supply (5) and grows on nearly 58 million acres of plantation and natural forest in the southeastern United States (6). Pine timber, including softwood and hardwood, is widely used in logs and lumber, as well as in the pulp and paper industry. Pine nuts are high in nutritional value. For instance, Stone pine (Pinus pineal) seeds have a protein content up to 31.6% of the total dry weight of the edible portion (7, 8). They also contain a considerable amount of vitamins A, B1, and B2, potassium, magnesium, and other minerals as well as dietary fiber (7, 9). Pine nuts are widely used for human consumption in Asia, Europe, and America. They are eaten either raw or roasted and have been used as ingredients in a variety of traditional recipes. Today, pine nuts are harvested in many regions of the northern hemisphere and are marketed globally as a gourmet product. The annual sales of pine nuts in the U.S. market total about 100 million dollars, 80-90% of which is imported, primarily from China where the most widely harvested species is Korean pine (Pinus koraiensis)(10), which is the most important species in today’s international pine nut trade. Realizing the importance and the lagging status of the conifer genomic study, compared with other agricultural crops, several genomic projects have been launched worldwide (3). In the United States, the Loblolly pine genome project started in 1999 * To whom correspondence should be addressed. Phone: 312-567- 3484. Fax: 312-567-3494. E-mail: zhangy@iit.edu. † Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology. ‡ Illinois Institute of Technology, National Center for Food Safety and Technology. § U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition. | U.S. Food and Drug Administration, National Center for Food Safety and Technology. J. Agric. Food Chem. 2008, 56, 8159–8165 8159 10.1021/jf801138q CCC: $40.75  2008 American Chemical Society Published on Web 08/09/2008