Phenylalanine:tyrosine ratio

The criteria for a biochemical diagnosis of untreated classic PKU are (1) a plasma phenylalanine level above 20 mg/dL (1.2mmol/L) (Figure 55-6, A) (2) a phenylalanine/ tyrosine ratio >3 (3) increased urinary levels of metabolites of phenylalanine (i.e., phenylpyruvic and 2-hydroxyphenyl-acetic acids) and (4) a normal concentration of the cofactor... 

The values for cystine and tyrosine were estimated on the basis of the methionine cystine and phenylalanine tyrosine ratios In human milk. 

Fig. 2. Phenylalanine/tyrosine ratios and phenylalanine hydroxylase activities in the liver of rats treated with -CPA and/or esculin. (Mean values SEM of four to six experiments.) p < 0.05.

Since L-tyrosine is the main precursor of melanins and a substrate of tyrosinase, the L-DOPA/L-tyrosine ratio could more accurately reflect tyrosinase activity than l-DOPA alone. In 1997, we developed two reversed-phase HPLC techniques, one with electrochemical detection to measure simultaneously l-DOPA, norepinephrine (NE), epinephrine (E), dopamine (DA), and DOPAC (3,4-dihydroxyphenyl acetic acid) (all compounds easily oxidizable between +0.15 and +0.50 V), and one with fluorimetric detection to measure L-tyrosine (and phenylalanine) on the same blood sample. 

Clinical studies on BH4 therapy have shown responders to BH, therapy to have improved phenylalanine to tyrosine ratios and less variability in blood phenylalanine concentration. 

Next to trypsin chymotrypsin is the most preferred proteolytic enzyme in sequencing. Its specificity is less absolute than that of trypsin. Primarily the bonds that follow phenylalanine, tyrosine and tryptophan are cleaved, but measurable hydrolysis takes place next to leucine and methionine residues as well. It is advisable, therefore, to determine in preliminary experiments the conditions (enzyme-substrate ratio, time, temperature) best suited for the formation of a few and well separable fragments. Occasionally also less specific enzymes, such as pepsin, papain or thermolysin find application in structure elucidation. For the hydrolysis of specific bonds new microbial proteases can be isolated. There are known prolidases and also enzymes which hydrolyze solely the bond which follows a pyroglutamyl residue and so on. 

Chace DH, Sherwin JE, and Hillman SL (1998) Use of phenylalanine-to-tyrosine ratio determined by tandem mass spectrometry to improve newborn screening for phenylketonuria of early discharge specimens collected in the first 24 hours. Clinical Chemistry 44 2405-2409. 

The aromatic amino acids each have two major absorption bands in the wavelength region between 200 and 300 nm (see reviews by Beaven and Holiday(13) and Wetlaufer(14). The lower energy band occurs near 280 nm for tryptophan, 277 nm for tyrosine, and 258 nm for phenylalanine, and the extinction coefficients at these wavelengths are in the ratio 27 7 l.(14) As a result of the spectral distributions and relative extinction coefficients of the aromatic amino acids, tryptophan generally dominates the absorption, fluorescence, and phosphorescence spectra of proteins that also contain either of the other two aromatic amino acids. 

The aromatic amino acids also have fluorescence emissions when excited by light in the UV range. Table Bl.3.3 gives the excitation wavelength, fluorescence emission wavelength, and quantum yield (Q) for tryptophan, tyrosine, and phenylalanine. The quantum yield is the ratio of photons emitted to photons absorbed. Typically, phenylalanine fluorescence is not detected in the presence of tyrosine and tryptophan due to low Q. Furthermore, tyrosine fluorescence is nearly completely quenched if the tyrosine residue is ionized or near an amino group, a carboxyl group, or a tryptophan residue (Teale, 1960 Freifelder, 1982). Therefore, tryptophan fluorescence is what is customarily measured. 

The limiting amino acid can be determined by taking the ratio of the amino acid present in the test protein and dividing it by the amount in the reference protein. The amino acid with the lowest ratio (test protein/reference) is the limiting amino acid. In this example, the single limiting amino acid is histidine (a dietary essential amino acid for young children). The lowest ratio is for the sum of phenylalanine and tyrosine. 

Modern UV spectrometers may offer the option of first and second-derivative UV spectroscopy and measurements in the 240-320 nm range using these techniques can be interpreted to determine the ratios of the various a romatic residues in peptides. Thus, tyrosine can be differentiated from tryptophan through first-derivative spectroscopy and phenylalanine can be differentiated from tyrosine and tryptophan through second-derivative spectroscopy (Miclo et al., 1995). 

Kwashiorkor Decrease in essential amino acids and tyrosine more than nonessential Ratio of essential to unessential amino acids helpful in diagnosis even with subclinical levels of protein deficiency abnormal phenylalanine tolerance test... 

Effect of coexisting species. The tolerable concentration ratios of foreign species on the determination of 10.0 pg of BSA in 10 mL of BSA-4MRASP system were as follows (the relative error less than 5 % fold) L-histidine, DL-aminoisovaleric acid, D-phenylalanine (60) L-leucine, DL-alanine (40) L-tryptophan, L-cysteine, L-tyrosine, DL-methionine (50) glycine (60) L-arginine (45) L-glutamic acid, L-lysine, L-methionine (70) DL-phenylalanine (100) ascorbic acid (200) Zn2+, Al3+ (20) K+, Fe2+ (30) Fe3+(5) Ca2+(20). 

Andrews [76] reported that plasteins were formed by an association of predominantly hydrophobic peptides via hydrophobic and possibly ionic bonding. Aso and coworkers [77,78] concluded that hydrophobic forces were a major factor in plastein chain assembly. They found that, compared with the substrate, the water-insoluble product contained smaller ratios of hydrophilic and larger ratios of hydrophobic amino acid residues. The results of Sukan and Andrews [58] showed that hydrophobic amino acids such as phenylalanine, leucine, isoleucine, tyrosine, valine, and proline were preferentially incorporated into plastein at the expense of hydrophilic amino acids. Also others have reported on a trend of preferential incorporation of hydrophobic amino acids into the protein product in enzyme-catalyzed reactions [46,60,79,80,81]. 

The absorption spectra of the proteins and (for comparison) the corresponding equimolar solutions (solution of tryptophan, tyrosine, and phenylalanine at the same ratio as they are contained in protein) are shown in Fig. 4. It is seen that the pwameters of the absorption bands of proteins do not coincide with the corresponding parameters for the equimolar solutions. 

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