Volume 55, Number 7, 2001 APPLIED SPECTROSCOPY 901 0003-7028 / 01 / 5507-0901$2.00 / 0 q 2001 Society for Applied Spectroscopy Vibrational Spectroscopic Study of L-Phenylalanine: Effect of pH S. OLSZTYNSKA, * M. KOMOROWSKA, L. VRIELYNCK, and N. DUPUY Institute of Physics, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland (S.O., M.K.); and Laboratoire de Spectrochimie Infrarouge et Raman (LASIR), UMR 8516 - CNRS, Bat. C5, Universite ´ des Sciences et Technologies de Lille, 59 655 Villeneuve d’Ascq, France (L.V., N.D.) Attenuated Total Reectance Fourier Transform Infrared (ATR- FTIR) spectra of L-phenylalanine in aqueous solution were recorded in the pH range 0–13. The vibrational molecular spectroscopic study of this amino-acid, a well-known component of human tissue, showed that L-phenylalanine undergoes structural changes under the inuence of pH. Characteristic bands of each ionic species were highlighted and the pKa values were determined from IR measure- ments by using an intensity ratio method. ATR-FTIR spectroscopy appears to be a powerful tool for obtaining valuable information on the building blocks of proteins and for measuring tissue damage in aqueous environments. Index Headings: ATR; FT-IR; Infrared; Vibrational spectroscopy; L-phenylalanine; Amino acid; pH; pKa. INTRODUCTION For many years vibrational spectroscopy techniques have been used widely and advantageously within such specialties as biochemistry, biophysics, and molecular biology, allowing the possibility of solving problems at the molecular level. 1 Infrared spectroscopy is often used for obtaining both structural and conformational information from biological samples, especially pro- teins 2–7 and amino acids. 8–12 Amino acids, which are the fundamental building units of proteins, are very important compounds as they take part in major metabolic processes, namely: growth, formation of new tissues, and biosynthesis of enzymes and hormones in the body. Their permeation through biological membranes depends on their pre- dominantly hydrophilic character. 13 Moreover, amino acids can be found in food, blood, urine, and some drugs where they can easily be identied and measured out by several analytical techniques. 14 Phenylalanine ( a-amino- b-phenyl-propionic acid) is one of the twenty biologically naturally occuring amino acids that can be found in proteins. It contains an amino group, a phenyl ring, and a carboxylic group. In aque- ous solution at neutral pH, the molecule adopts a zwit- terionic structure (Fig. 1b). If the zwitterion does not exist in the gas phase, it can nevertheless appear in KBr matrix (solid solvent) prepared by the dissolution, spray, and deposition method (called DSD process), a new infrared sampling technique developed by Cao and Fischer. 12 With this method, the largely monomeric zwitterion is stabilized by matrix eld effects. Phenylalanine, as with many other amino acids, is not easily studied by vibrational spectroscopy tech- Received 27 June 2000; accepted 2 February 2001. * Author to whom correspondence should be sent. niques because the infrared and Raman spectra of ami- no acids in aqueous solution are usually broad, over- lapped, or incomplete as a result of strong solvent ab- sorption and solute-solvent interactions. Very few in- frared investigations have been reported until now to our knowledge in the literature; furthermore, these ar- ticles generally deal with phenylalanine in the solid state. 12,14 On the other hand, many NMR studies have already been published about kinetics of phenylalanine transport at the human blood-brain barrier of patients with phenylketonuria. 15–19 These results show that the aromatic amino acids, especially phenylalanine, are re- quired for structural stability during electron transport. The role of phenylalanine in determining the membrane position, dynamics, and free energy of a peptide was also examined. 20 In this paper, the effect of pH on tissue model-like L-phenylalanine was investigated by means of ATR- FTIR spectroscopy. This infrared technique was chosen because it allows the identication of amino acids in different states, such as powders and in aqueous solu- tions. 21–23 The vibrational spectrum of L -phenylalanine was rst measured in water solution at 0.1 M and com- pared with the spectrum of the crystalline form. After- wards, the ATR-FTIR spectra in aqueous solution were analyzed at different pH levels (from 0 to 13) and the p K a values of the two ionizable functions were deter- mined by means of a band intensity ratio method. MATERIALS AND METHODS Reagents. L-phenylalanine solid compound was pur- chased from the Sigma Chemical Co. (St. Quentin Fal- lavier, France) with a purity not less than 99% and was used as received without further purication. Sampling. In the rst part of the infrared study, the solutions were prepared by dissolving solid L-phenyl- alanine in distilled water and heavy water at a concen- tration of 0.1 mol L 2 1 (pH 5 6.25). In the second part of the study, aqueous solutions of L-phenylalanine were prepared as follows: the solid compound was rst dis- solved in HCl aq (1 mol L 2 1 ) or NaOH aq (1 mol L 21 ) at a concentration of 0.5 mol L 2 1 . Afterwards, from these two initial solutions of L -phenylalanine, controlled vol- umes of NaOH aq or HCl aq were carefully added to set higher or lower pH values in order to set the same nal concentration in amino acid (0.25 mol L 21 ). A process of gelication was observed starting at pH 5 2.5 and occurring until pH 5 8.76 (although no gel was formed in distilled water) possibly due to the formation of aqueous NaCl salt.