Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride Neelesh Singh & Reetesh Kumar & M. V. Jagannadham & Arvind M. Kayastha Received: 31 December 2012 / Accepted: 19 February 2013 / Published online: 28 February 2013 # Springer Science+Business Media New York 2013 Abstract Physiologically as well as industrially, α-galactosidases are very important en- zymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluores- cence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α + β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed. Keywords α-Galactosidase . Fluorescence . Circular dichroism . Molten globule . Unfolding . Guanidine hydrochloride Abbreviations GuHCl Guanidine hydrochloride CD Circular dichroism MG Molten globule RFOs Raffinose family oligosaccharides pNPGal p-Nitrophenylα-D-galactopyranoside ANS 8-Anilino-1-napthalenesulfonic acid Appl Biochem Biotechnol (2013) 169:2315–2325 DOI 10.1007/s12010-013-0163-9 N. Singh : A. M. Kayastha (*) School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India e-mail: kayasthabhu@gmail.com R. Kumar : M. V. Jagannadham Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India