Effects of Varied Tyrosine Concentrations on FEEM and SFS of Constant Tryptophan concentration

In recent years, the 3D fluorescence excitation emission spectrum has been widely applied into the analysis of the properties of soluble organic matter in water bodies, but there is very little research on the fluorescence properties of protein residues in water quality detection by Fluorescence Excitation Emission Matrix (FEEM), and synchronous fluorescence excitation emission spectra (SFS). The characteristics of luminescent of amino acids, including tryptophan, tyrosine and phenylalanine, where tryptophan and tyrosine fluorescence are relatively strongest, and the effects of tyrosine on the fluorescence properties of tryptophan are seldom discussed in different mixing ratios for both amino acids. The fluorescent peak positions and intensities of the three amino acids are related to the angle size of conjugate plane, N hybrids and hydroxyls on benzene rings. When the ratio of tyrosine to tryptophan content is less than 150:100, tryptophan will quench the fluorescent peak of the tyrosine. Constant tryptophan content, tyrosine at excitation wavelength of 228 nm, its concentration changes with the corresponding fluorescence intensity has a good linear relationship; however, at 276-278 nm of tyrosine, the varied concentrations and fluorescence intensities are opposite.


Introduction
The soluble organic matter, 30-40% of total organic pollutants in city sewage [1], is the major target in sewage treatment. monitoring the amount and characteristic of organic matter is always an important item in research of sewage treatment. Conventional organic pollutant parameters like chemical oxygen demands(COD), total organic carbons(TOC) can be only used to indicate the amount of organic matter but not the characteristics and ingredients of organic matter, which also need more water sample volume and operating time. In recent years, highly sensitive and using little sample volume, the 3D fluorescence excitation emission spectrum has been widely applied into the analysis of the properties of soluble organic matter in water bodies [2,3]. It reported that as proteins, humus and grease in life sewage possess fluorescence, 3D fluorescence excitation emission spectrum can be developed as a new method of indicating organic matter [4,5]. Chen et al divided the 3D fluorescence excitation emission spectrum of city sewage into five fingerprint areas. Otherwise, the area for protein amino acid residues was seldom researched in more detail, especially for the first and second area belonging to themselves and the fourth area belonging to solute products of microbial degradation [9,10].
Currently, most studies on the fluorescence properties of amino acids in water are those of molecular structure and fluorescence properties, the effects of fluorescent additives on the fluorescence properties of amino acids, correlation between fluorescence properties and COD, the difference of the fluorescence properties of amino acid residue from different sources. However, the fluorescence spectrum of single amino acid solution has been seldom studied, especially for that of amino acid mixture, and the mutual effects of coexisting of two type of amino acid residues on fluorescence properties have been little reported [11,12]. Therefore, this paper will get on the research of 3D fluorescence excitation emission spectrum for the artificial solutions of tryptophan, tyrosine and phenylalanine, and the mixtures of tryptophan and tyrosine will be detected to discuss the mutual effects of EEFM and SFS.

Preparation of reagents and samples
Tree kinds of fluorescent samples: tryptophan, tyrosine and phenylalanine with a purity of more than 99% were bought from Sigma company. 1000 mg/L each sample solution was prepared by weighting the sample accurately to 0.0001 g and using free-ion water to dissolved it completely. Other 5 mg/L, 0.1 mg/L sample solutions were prepared by dilution from 1000 mg/L.

Instrument and experimental condition
Fluorescence spectrometer(F-4500, Hitachi, Japan)equipped with a 150 w xenon light and a 700 V photomultiplier tube detector was used to scan samples at a single wavelength or also at full wavelength to obtain their 3D spectrum

Results and discussions
3.1 Characteristic analysis of 3D fluorescence excitation emission spectrum 50 mg/L tryptophan, tyrosine and phenylalanine solutions were scanned individually by 3D fluorescence spectrometer and the fluorescent intensities all exceeded the upper limit of this instrument. Therefore, the lower concentration (0.1 mg/L) diluted solutions were detected to obtain their spectrum ( Figure 1). The results of comparison for target peak intensity of three amino acids indicate that tyrosine is higher than tryptophan, and phenylalanine is the lowest. In order to view the spectrum of phenylalanine more clearly, 5 mg/L phenylalanine solution was scanned to obtain Figure 1 C-2. The positions of two target peaks for their spectrums were Ex/Em: 226/301nm(1270) and 214/304 nm(1212) for 0.1 mg/L tyrosine solution, Ex/Em: 222/349 nm (1328) and 278/352 nm (2585) for 0.1 mg/L tryptophan solution, and Ex/Em: 214/280 nm (1502) and 258/283 nm (1225) for 0.5 mg/L phenylalanine solution. The two target peaks for every one of three amino acids can be attributed to two conjugated plane structures in their molecular structure including an aromatic ring and a carboxyl acid group. which are not coplanar each other [15]. Because of the more rigid structure of benzene ring adjacent with N-hetercyclo ring for Tryptophan, the fluorescent intensity is more than the other two and the peak emission wavelength is red shift. Comparing with Phenylalanine, the fluorescent intensity and wavelength of Tyrosine is higher than Phenylalanine due to the phenol group which makes the conjugated length longer than benzene ring. The intensity of the peak at 210-250 nm wavelength are much more than that at 250-300 nm wavelength. The single peak of spectrum for tryptophan happens at 250-300 nm wavelength, which may be a combined peak from two peaks at near position. Because the intensity of the peak of spectrum of 0.1 mg/L Phenylalanine is too weak, we explore the spectrum of 0.5 mg/L Phenylalanine. Two peaks of   [16].

Characteristic analysis of simultaneous fluorescent spectrum of Tyrosine and Tryptophan
Molecular structures of the proteins in water generally have the residue of Tyrosine or Tryptophan. Therefore, the fluorescent spectra of mixtures of Tyrosine and Tryptophan were explored to understand the change of spectrum before and after mixing them. Figure 3.  According to the peaks occurring at Ex/Em: 226/301nm and 214/304 nm for the spectrum of tyrosine, we find that when the concentration ratio for tyrosine and tryptophan is below 150/100 nm, the target peaks of the spectrum of tyrosine disappear ( Figure 3A,3B) [17]. This can be attributed that tryptophan quenches the fluorescence of tyrosine. The target peaks occurring at Ex/Em: 222/349 nm and 278/352 nm for the spectrum of tryptophan are not affected by tyrosine in the spectrum of the mixture. The target peaks of tyrosine in the spectrum of the mixture appear more and more clearly by increasing the concentration ratio of tyrosine ( Figure 3C, 3D, 3E and 3F).    2. The fluorescent intensity and wavelength of Tyrosine is higher than Phenylalanine due to the phenol group which has longer conjugated length than benzene ring.
3. when the concentration ratio for tyrosine and tryptophan is below 150/100, tryptophan quenches the fluorescence of tyrosine.
4. The fluorescent intensity and concentration for tyrosine have a positive linear relationship at excited wavelength 228 nm and at excited wavelength 276-278 nm, the fluorescent intensity little decrease by enhancing concentration of tyrosine.