Purification of a PHA-Like Chitin-binding Protein from Acacia farnesiana Seeds: A Time-dependent Oligomerization Protein

• Published in: Applied biochemistry and biotechnology Vol. 150; no. 1; pp. 97 - 111
• Main Authors: Santi-Gadelha, T, Rocha, B.A.M, Aragão, K.S, Marinho, E.S, Gadelha, C.A.A, Toyama, M.H, Pinto, V.P.T, Nagano, C.S, Delatorre, P
• Format: Journal Article
• Language: English
• Published: New York Humana Press Inc 01-07-2008; Springer; Springer Nature B.V
• Subjects: Acacia - chemistry; Acacia farnesiana; agglutination tests; albumins; Amino Acid Sequence; amino acid sequences; Biochemistry; Biological and medical sciences; Biotechnology; Carbohydrates; chemical constituents of plants; Chemistry; Chemistry and Materials Science; Chitin; Chitin - chemistry; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Dolichos biflorus; Erythrocytes; Fabaceae; Flowers & plants; Fundamental and applied biological sciences. Psychology; gel electrophoresis; humans; ion exchange chromatography; lectins; Liquid chromatography; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Phaseolus vulgaris; Plant Lectins - analysis; Plant Lectins - chemistry; Plant Lectins - isolation & purification; plant proteins; Proteins; purification; rabbits; Seeds; Seeds - chemistry; Sequence Alignment; Sequence Analysis, Protein; Sodium chloride; Studies; Tandem Mass Spectrometry; Tandem mass spectrometry; Lectin-like protein; Purification; Oligomerization; Lectin; Binding protein; Leguminosae; Dicotyledones; Mass spectrometry MS/MS; Acacia farnesiana· Lectin-like protein, Purification, Oligomerization; Angiospermae; Acacia; Spermatophyta; Chitin
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-008-8144-0

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI = 4.0 +/- 0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.