Tyrosine spectrophotometric determination

Price, M.L. and Butler, L.G., Rapid visual estimation and spectrophotometric determination of tannin content of sorghum grain, J. Agric. Food Chem., 25, 1268, 1277, 1977. Folin, O. and Ciocalteu, V., On tyrosine and tryptophan determinations in proteins, J. Biol. Chem., 27, 627, 1927. 

Application and Principle This procedure is used to determine the proteolytic activity, expressed as hemoglobin units on the tyrosine basis (HUT), of preparations derived from Aspergillus oryzae var. and Aspergillus niger var., and it may be used to determine the activity of other proteases at pH 4.7. The test is based on the 30-min enzymatic hydrolysis of a hemoglobin substrate at pH 4.7 and 40°. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration. The quantity of solubilized hemoglobin in the filtrate is determined spectrophotometrically. 

Table I gives the amino acid composition of bovine chymotrypsinogen A. Quite similar results for most amino acids have been obtained for this protein in two laboratories by the usual chromatographic technique in its manual or automatic (83) form involving all the necessary corrections and extrapolations to zero time. Cystine has been determined as cysteic acid after pcrformic acid oxidation. Values of tryptophan are probably less satisfactory sin( e they are derived from spectrophotometric or microbiological determinations. The protein contains only 2 histidines, 2 methionines, 4 arginines, 4 tyrosines, and 5 cystines per mole.

The potential advantages of selective nitration of tyrosyl residues in native proteins are numerous. The reaction is performed under mild conditions, giving rise to a 3-nitrotyrosyl derivative (pK 7), which in the acid form absorbs intensely at 350 nm. Hence, the nitrotyrosine content may be readily determined spectrophotometrically, as well as by amino acid analysis ( 2.2.3). The absorption spectrum of 3-nitro-tyrosine is highly sensitive to solvent polarity and exhibits significant optical activity in the long wavelength absorption band. Consequently, nitrotyrosyl residues can be utilized as indicators of conformational change, or of interactions of proteins with other macromolecules or small molecules (e.g. Kirschner and Schachman 1973). Any perturbation in the pK of nitrotyrosyl residues is readily determined spectrophotometrically. 

Chlorine dioxide is determined from its absorbance as the coloured product (Xmax = 490 nm) of its reaction with tyrosine [48]. Chlorine dioxide has been found to reduce the absorbance of Acid Chrome Violet K [49]. Spectrophotometric methods of determining CIO2 with the use of Chlorophenol Red have been proposed [50,51]. A scheme for analysing mixtures of Cf, CIO, CIO2, ClO.i, CIO4 , and CIO2 using spectrophotometric and other methods has been devised [52]. 

All other available methods for determining serum total proteins have been discussed above in relation to albumin, and their limitations indicated. All methods suffer from differences in the individual protein s contribution of the parameter being measured. However, the tyrosine (Folin-Ciocalteu) and arginine (Sakaguchi) equivalence methods (S7) and the near-ultraviolet spectrophotometric methods are more severely affected in this respect than other methods, and are therefore unreliable for total serum protein analysis. 

Apart from this the interest and application of ultraviolet spectra of proteins are analytical. On a microscale the absorption spectrum may be the simplest and best evidence for the recognition of a protein. It is possible that, with care, it will be the best means of obtaining an estimate of tyrosine and tryptophan in a protein. The instability of tryptophan under the conditions required for protein hydrolysis gives weight in favor of a method such as the spectrophotometric which allows a direct determination of tryptophan to be made (on a protein) without hydrolysis. 

There are several recorded determinations of the absorption curves of the aromatic amino-acids. Most of these were obtained with photographic methods of spectrophotometry which have been superceded by more accurate photoelectric methods. It will be shown that in the spectrophotometric analysis of tyrosine and tryptophan in proteins, the photometric error is magnified in the final estimate of tyrosine and tryptophan contents. This fact is inevitably bound up with the form of the equations of mixture analysis. It is therefore important that the absorption constants be measured as accurately as possible. 

The difference absorption spectra of hen and turkey lysozymes in the alkaline pH region had three maxima at around 245, 292, and 300 nm and had no isosbestic points.The ratio of the extinction difference at 245 nm to that at 295 nm changed with pH. These spectral features are quite different from those observed when only L-tyrosyl residues are ionized, and it was impossible to determine precisely the pK values of the L-tyrosyl residues in lysozyme by spectrophotometric titration. A time-dependent spectral change was observed above about pH 12. This is not due to exposure of a buried L-tyrosyl residue on alkali denaturation. The disulphide bonds and the peptide bonds in the lysozyme molecule were cleaved by alkali above about pH 11. The intrinsic pK value of L-tyrosine-23 of hen lysozyme was determined to be 10.24 (apparent pK 9.8) at 0.1 ionic strength and 25 C from the c.d. titration data. Comparison of the c.d. 

The spectrophotometric method has been used with reasonable success, but great difficulties can be encountered in the determination of tryptophan by the spectrophotometric method when this amino acid is present in the protein at a low level or when the tyrosine content is high. 

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