ELSEVIER Biochimica et Biophysica Acta 1289 (1996) 51-56 BB, Biochi]ic~a et Biophysica A~.ta Studies on metal induced conformation changes in a peripheral blood lymphocyte lectin Rajeev Kayestha, Sumati, Krishnan Hajela * School of Life Sciences, Devi Ahilya Vishwavidyalaya, Vigyan Bhawan, Khandwa Road Campus, lndore-452001, India Received 7 June 1995; revised 5 September 1995; accepted 11 September 1995 Abstract A Ca 2+ dependent glucose specific lectin was isolated from goat peripheral blood lymphocytes by affinity chromatography on N-acetyl D-glucosamine agarose gel. Since the lectin binding to carbohydrate ligands was metal-dependent, it was important to study the divalent metal ion induced conformational changes in the lectin. The conformational changes were studied by absorption, fluorescence and circular dichroism spectroscopy techniques. Binding of Ca 2÷, Mn 2+ or Mg 2÷ results in shift in ultraviolet absorption maxima from 281 to 298 nm (red shift). A major increase in absorbance at 245 nm is also exhibited. Binding of Ca 2÷ and Mn 2+ resulted in decrease in intrinsic fluorescence emission maxima with shift from 355.2 nm to 342.4 nm (blue shift). These shifts could be reversed on addition of EDTA. A double reciprocal analysis of fluorescence quenching data suggest that Ca 2+ and Mn 2+ interact with a single class of binding site with an apparent k d of 1.50 + 0.37 /zM and 1.25 ___ 0.25 /xM, respectively. These data clearly indicate that occupancy of metal binding sites on goat peripheral blood lymphocyte lectin induces a gross conformational change sequestering aromatic amino acids into a hydrophobic environment. These findings were further supported by circular dichrosim spectrum which showed a massive alteration in the presence of Ca 2÷. 1. Introduction A variety of important functions are carried out by calcium ions in biological systems [1,2]. Certain calcium binding proteins play a critical role in the execution of these functions. In some proteins (like trypsin), the cal- cium ion is apparently structural, stabilizing a local region of the structure, not participating directly in catalyst or regulatory process [3]. In others (like Staphylococcusnu- clease), it is directly involved in aspects of catalysis [3]. In the third group of proteins (the calmodulin superfamily), calcium binding triggers a conformational change thus playing a regulatory or signalling role [4,5]. Apart from calmodulin superfamily another superfamily exhibiting Ca 2+ dependent activity is the C type (Ca 2+ dependent) carbohydrate recognition domain (CRD) superfamily [6] with constituent members having a general property of Abbreviations: CRD, carbohydrate recognition domain; GPBL, goat peripheral blood lymphoc~(tes; PMSF, phenyl methyl sulfonyl fluoride; Tris buffer, 10 mM Tris HCI, pH 7.5 containing 150 mM NaC1 and 0.1% sodium deoxycholate * Corresponding author. Fax: +91 731 472793. 0304-4165/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0304-41 65(95)001 35-2 interacting with carbohydrate structure in a Ca 2+ depen- dent manner [7,8]. The C type (Ca 2+ dependent) animal lectins are the most well known examples of this super- family. We have earlier isolated a Ca 2÷ dependent (C type) glucose specific lectin from goat peripheral blood lymphocyte (GPBL) [9]. As presence of Ca 2÷ was essen- tial for the lectin activity it was obvious that Ca 2+ plays a important role in modulating the structure and function of this lectin as well. It was speculated that binding of Ca 2÷ might produce a conformational change in lectin molecule thereby converting in from an inactive to active form. Role of other divalent metal ions in replacing Ca 2÷ was also to be investigated. The present paper gives evidence based on various spectroscopic techniques indicating that binding of divalent metal ions produces gross conformational changes in a lymphocyte lectin. 2. Materials and methods 2.1. Materials Histopaque-1077, PMSF, Triton X-100 (TX-100), Sephadex G25, sodium deoxycholate and N-acetyl o-glu-