Thermodynamics of interaction of caffeic acid and quinic acid with multisubunit proteins
Introduction
Polyphenolic compounds are secondary plant metabolites found in numerous plant species. Among polyphenols, chlorogenic acid (CGA) is an important phenolic acid from a physiological point of view and, upon hydrolysis, it yields caffeic acid (CA) and quinic acid (QA). CA is also called 3,4-dihydroxy cinnamic acid. It has two phenolic groups and therefore acts as an antioxidant [1], [2], [3], [4], [5]. CA is also reported to be an anticarcinogen [6]. On the other hand, CA binds to the enzymes of physiological significance and inhibits their functional role [7], [8], [9], [10]. As an ortho-diphenol, CA in alkaline pH is converted to O-quinone, which can then covalently link with the nucleophilic groups of the protein molecule [11], [12]. Polyphenols decrease the digestibility of the protein molecules [13]. Despite the role of CA and QA in a wide variety of biological processes, no detailed study is reported on the nature of the interaction of CA and QA with proteins.
Helianthinin is a major protein fraction from Sunflower seeds [14]. It is a multisubunit protein with six acidic and six basic subunits. Each acidic and basic subunit is linked by disulphide bridges to form a monomeric unit. Six such monomers associate non-covalently to form a hexameric protein. Its dissociation and denaturation behaviour is studied and a model proposed [14].
Interaction of polyphenols with the proteins was studied earlier [13], [15], [16], [17]. Hydrophobic interaction is reported to be the driving force for this interaction [15], [16], [17]. Enthalpy–entropy compensation is reported to be present in protein–polyphenol interaction [7]. Interaction of polyphenols with multisubunit proteins has not been studied in detail. Moreover, the interaction mechanism of CA, a polyphenol, and QA, a non-phenolic molecule with proteins, is not clear. Thus, in the present study, the interaction of CA and QA with multisubunit protein helianthinin from plant systems and human haemoglobin (Hb) from mammals is studied to understand the effect of interaction of the presented ligands and the stability of these two proteins.
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Materials
CA, QA, Hb, N-bromosuccinimide, succinic anhydride, N-acetyl-l-tyrosine ethyl ester and N-acetyl-l-tryptophan ethyl ester were procured from Sigma Chemical Company (St. Louis, MO, USA). Sodium dihydrogen orthophosphate, disodium hydrogen phosphate and ammonium sulphate were from S.D. Fine Chemicals (Boisar, India). Dialysis tubing was obtained from Thomas Scientific Co. (Philadelphia, USA). Quartz double-distilled water was used in all experiments.
Preparation of Sunflower seed flour
The flour of Sunflower seeds of the Morden
Results
The results are analysed for interaction of CA with helianthinin and QA with helianthinin as well as with mHb independently, in terms of the binding, thermal denaturation properties and DSC profiles, to bring about the differences and commonalities to understand the mechanism of interaction of these ligands with both multisubunit proteins.
Discussion
After ingestion of food materials containing polyphenols such as CGA and its hydrolytic products, CA and QA enter into the blood stream after absorption through intestinal villi and these free ligands interact with many proteins. Our earlier studies on these ligands with human serum albumin [16], which is a single polypeptide chain, indicate the predominance of hydrophobic interaction.
Thus, this study would enlarge the database for understanding the mechanism and thermodynamics of interaction
Acknowledgements
P.S. and R.P.K. are supported by a fellowship from CSIR, New Delhi, India.
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