543 Journal of Protein Chemistry, Vol. 22, No. 6, August 2003 (© 2003) 0277-8033/03/0800-0543/0 © 2003 Plenum Publishing Corporation Characterization of a Proteinase Inhibitor from Cajanus cajan (L.) Soghra Khatun Haq 1 and Rizwan Hasan Khan 1,2 Received March 14, 2003 A protein proteinase inhibitor (PI) has been purified from pigeonpea Cajanus cajan (L.) PUSA 33 variety by acetic-acid precipitation, salt fractionation and chromatography on a DEAE-Cellulose column. The content of inhibitor was found to be 15 mg/20 g dry weight of pulse. The molecular weight of the inhibitor as determined by SDS-PAGE under reducing conditions was found to be about 14,000. It showed inhibitory activity toward proteolytic enzymes belonging to the serine pro- tease group, namely trypsin and -chymotrypsin. The inhibitory activity was stable over a wide range of pH and temperatures. Estimation of sulfhydryl groups yielded one free cysteine and at least two disulfide linkages. N-terminal sequence homology suggests that it belongs to the Kunitz inhibitor family. Structural analysis by circular dichroism shows that the inhibitor possesses a largely disordered structure. KEY WORDS: Cajanus cajan; circular dichroism; fluorescence quenching; Kunitz inhibitor; N-terminal sequence; proteinase inhibitor. 1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India. 2 To whom correspondence should be addressed. E-mail: rizwanhkhan@ hotmail.com 1. INTRODUCTION Proteinase inhibitors (PIs) comprise one of the most abundant classes of proteins in plants (Ussuf et al., 2001). Most storage organs such as seeds and tubers con- tain 1 to 10% of their total proteins as PIs, which inhibit different types of enzymes (Ryan, 1981; Pearce et al., 1982). These proteins have diverse biochemical func- tions, some of which include elimination of unwanted proteolysis (Laskowski and Kato, 1980), inhibition of in- traerythrocytic development of Plasmodium falciparum (Rockett et al., 1990), suppression of in vitro and in vivo replication of retroviruses (Vonderfecht et al., 1988) and inhibition of growth of transformed cells (Schnebli and Burger, 1972; Chou et al., 1974). The plant proteinase inhibitors of serine proteinases play a dominant role in natural plant defence and infection processes (Ramasarma et al., 1994). C. cajan (L.) Millsp. is one of the oldest food crops and ranks fifth in importance among edible legumes of the world (Salunkhe et al., 1986). It is important in human nutrition as a rich source of dietary protein (Singh et al., 1984). However, preharvest damage due to insect pests on developing seed and postharvest losses due to storage pests are severe (Reed and Lateef, 1990). Consequently, the amylase inhibitors (AIs) and pro- teinase inhibitors (PIs) have gained attention as possible tools of natural and engineered resistance against pests and pathogens (Ryan, 1990; Giri and Kachole, 1998). Besides this, increased exposure to dietary protease in- hibitors has been shown to protect against some chemically induced animal tumors (Weed et al., 1985; Mokhtar et al., 1988) and it has been suggested that protection against cancer may be achieved through this avenue (Schelp and Pongpaew, 1988). The legume proteinase inhibitors are classified into two main groups according to their size and cystine con- tent. One group of inhibitors, the Bowman-Birk type, has molecular weights of around 8000 and relatively high cystine content. The other main category of inhibitors, the 3 Abbreviations: BAPNA, N  -Benzoyl-L-Arg-p-nitroanilide; DTT, dithiothreitol; GPNA, Glutaryl-Gly-Gly-Phe -naphthylamide; NATA, N-acetyl L-tryptophanamide; PI, proteinase inhibitor; SDS- PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.