Fluorescence Study of Sinapic Acid Interaction with Bovine Serum Albumin and Egg Albumin
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Fluorescence Study of Sinapic Acid Interaction with Bovine Serum Albumin and Egg Albumin Bogdan Smyk Received December 4, 2002; revised April 14, 2003; accepted April 14, 2003
The mechanism of interaction of protein with compounds used for preparation of matrices for matrixassisted laser desorption ionization–mass spectrometry (MALDI-MS) methods is unknown. This paper reports the investigation of this mechanism for sinapic acid and bovine serum albumin and egg albumin. To examine these interactions in water a fluorescence method was applied. Sinapic acid can exist in three different forms, depending on pH: undissociated and with one or two deprotonated groups. pKas of these states are: 4.47 for the COOH group and 9.21 for the OH group [1]. Therefore the interactions were examined at pH: 2.0, 6.4, and 10.5. The results show that sinapic acid at pH 10.5, being a bivalent anion, does not form any complex with these two proteins. At pH 2.0, sinapic acid, being undissociated, interacts weakly with egg albumin. Sinapic acid does not interact with bovine serum albumin at this pH. At pH 6.4, sinapic acid interacts only with bovine serum albumin. Parameters of the sinapic acid and bovine serum albumin complex were calculated based on the theory of multiple equlibria: the total number of binding sites, N 15; the binding constant, K 600 M1; and the Hill’s coefficient, j 0.97. These parameters indicate (but not definitively because a large saturation was not obtained) that this is a simple binding of sinapic acid to bovine serum albumin with the binding sites of the same type.
KEY WORDS: Sinapic acid; flourescence; ligand-protein interaction; pH.
the matrix with protein. The interaction of proteins with compounds used for matrix formation in solution and during the process of crystallization affects the protein distribution in the matrix. It seems that the complex should not dissociate during the process of drying and crystallization because lyophilized proteins exhibit “pH memory,” that is, their behavior in the solid form corresponds to the pH of the aqueous solution from which they were freeze dried [5]. The pKa values in the aqueous and lyophilized states for each compound containing groups that can dissociate in protein were found to be similar [5]. However, it is necessary to be cautions, because local pH may be dramatically changed depending on the buffer used [6]. In the dried and frozen states, proteins undergo deterioration that may be both chemical and conformational [7]. Their secondary and quaternary
INTRODUCTION Since 1988, work has been developed on matrixassisted laser desorption ionization–mass spectrometry (MALDI-MS), a new method for mass spectrometry of proteins. The search continues for matrix compounds. Cinnamic acids and benzoic acids seem to be good candidates for matrix-forming compounds. Sinapic acid (SA) has been used as a one of the main compounds for matrix formation [2–4]. To prepare a matrix with protein, the protein solute is added to the matrix compound solution, and then some aliquot of
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