Purification of a Zinc Binding Protein from Xylem of Citrus jambhiri

sporamin ABSTRACT. Zinc in xylem and phloem of the citrus rootstock, rough lemon (Citrus jambhiri (L.)) was associated with a Zn- binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 N NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors. samples were collected from healthy or blight affected trees of Citrus jambhiri grown in a quarantine greenhouse facility on the campus of University of Arizona, Tucson, during 1996. Xylem evacuate was collected by applying 4 mL of 10 mM Tris-Cl, pH 7.4 to one end of a 10 cm root piece >1 cm diameter while a vacuum was pulled at the other end. Bark was removed from the root piece down to the cambium before evacuation. Before purification, xylem evacuate was stored at -80 °C. Phloem tissue was sampled as a bark patch taken 15 to 30 cm above the soil line. Contaminants on the bark surface were removed by scraping away the brown-green outer layer to a 5 × 10 cm rectangle of clean whitish phloem tissue. The patch was scored through the scraped bark to the wood and separated from the tree at the cambial layer. The samples were taken from convex surfaces of healthy and blight-affected trees in active growth. ISOLATION AND PURIFICATION OF CVZBP. To quantitate recov- ery of CVZBP, three independent purifications were performed. Three separate xylem evacuates (Derrick et al., 1990) were lyophilized and adjusted to 1 mL each with deionized water. Xylem evacuate was fractionated by ion exchange chromatogra- phy (IEC) (Taylor et al., 1996) over quaternary aminoethyl Sepharose (QAE) (Pharmacia, Piscataway, N.J.). Total Zn was determined for aliquots of each fraction by determination of A213.9 by atomic absorption spectrometry (model 3100, Perkin-Elmer, Shelton, Conn.) and for 254 nm absorbance (A254) by ultraviolet (UV)- spectrophotometry. Fractions with elevations of A254 and coincident elevated Zn were pooled and the volume reduced in a centrifugal vacuum concentrator (model RC 10. 10; Jouan, Win- chester, Va.). Concentrated Zn-containing eluant from QAE chromatography was separated using a 2.5 × 14 cm gel filtration (GF) column of Bio-Gel P-30 (Bio-Rad, Hercules, Calif.) in 10 mM Tris-Cl buffer, pH 8.0 (4 °C) in 3 mL fractions. Fractions within the peaks of A254 and elevated Zn content were pooled, trichloroacetic acid precipitated (Deutscher, 1990), and further purified by sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) (Laemmli, 1970). For all isolation steps, total protein was determined at A595 with Quantigold (Diversified Biotech, Newton Centre, Mass.) or with the Bradford assay (Bio- Rad), depending upon protein concentration with each stage of purification.