Isolation and purification of phytocystatin from almond: Biochemical, biophysical, and immunological characterization

Abstract

It is well known that fruit nuts contain wide variety of flavonoids and various proteins, consumption of which has been associated with the reduced risk of chronic diseases. Cystatins, a family of cysteine proteinase inhibitors, ubiquitously present in all cells serve various important and critical physiological functions. In this study a phytocystatin with molecular mass of 63.4 kDa was purified to homogeneity by a three-step process including ammonium sulfate fractionation (50–70%), acetone precipitation, and gel filtration chromatography on Sephacryl S100-HR column. The purified inhibitor migrated as single band under native and SDS-PAGE. The Ki values for purified inhibitor with papain, ficin, and bromelain were found to be 45.45, 83.33, and 90.9 nM, respectively, suggesting higher affinity of the inhibitor for papain as compared to ficin and bromelain. Phytocystatin was stable in broad pH and temperature range. Purified cystatin appeared to be antigenic as observed in western blot analysis. ITC assay data show a binding stoichiometry of 0.870 ± 0.03 sites for cystatin and papain interaction which indicated that cystatin is surrounded by nearly one papain molecule. FTIR, UV, and fluorescence studies showed significant conformational changes on cystatin–papain complex formation. Purified cystatin was found to possess 36.8% α-helical content as observed by CD spectroscopy.

Keywords:

• phytocystatin
• almond
• spectroscopy
• kinetics
• CD
• FTIR
• ITC

Public Interest Statement

Plant cystatins or phytocystatins are the second most studied class of protease inhibitors. They are cysteine (thiol) protease inhibitors that interact with the active site of target proteases and block access to their protein substrates. Since the publication of a first full paper reporting their existence, phytocystatins have shown wide application in pharmaceutical, agricultural, and industrial field. These plant derived inhibitors are also gaining wide consideration in drug designing and engineering GM crops. Owing to the great significance and importance of these inhibitors, it is essential to explore and isolate them from various plant sources. In the present study, a phytocystatin has been isolated and purified from almond which is a source of high-quality protein with great nutritive value. Almond cystatin was purified to homogeneity by a three-step process including ammonium sulfate precipitation and gel filtration chromatography. Purified inhibitor was further characterized using various biophysical and biochemical techniques.

Competing Interests

The authors declare no competing interest.

Acknowledgment

Facilities provided by Aligarh Muslim University are gratefully acknowledged. We are grateful to SAP-DRS-III and UGC-BSR program for their generous research support.

Additional information

Funding

Funding. The authors received no direct funding for this research.

Notes on contributors

Azad Alam Siddiqui

Bilqees Bano is an active member of the Department of Biochemistry, Faculty of Life Sciences Aligarh Muslim University, Aligarh. She has received several fellowships (at pre-PhD level from CSIR-JRF, SRF; at post-PhD level from CSIR-PDF Award and Pool Officer Fellowship; Commonwealth Academic Staff Fellowship—UK; Indo-US Fulbright fellowship—USA; NIH Fellowship Award—USA). She has 34 years’ research experience in the fields of proteins, enzymes, and clinical biochemistry. Bano’s current interest is in cysteine protease inhibitors with particular reference to their amyloid formation and evaluation of potential candidates for their anti-amyloidogenic properties.