Tyrosine purification

All the filtrations in the purification of tyrosine, except possibly the last, are best done on a 20-cm. Buchner funnel. Whenever charcoal is used, kieselguhr may be employed to obtain a clear filtrate. 

Detection and quantification of protein by measuring absorbency at 280 nm is perhaps the simplest such method. This approach is based on the fact that the side chains of the amino acids tyrosine and tryptophan absorb at this wavelength. The method is popular, as it is fast, easy to perform and is non-destructive to the sample. However, it is a relatively insensitive technique, and identical concentrations of different proteins will yield different absorbance values if their content of tyrosine and tryptophan vary to any significant extent. Hence, this method is rarely used to determine the protein concentration of the final product, but it is routinely used during downstream processing to detect protein elution off chromatographic columns, and hence track the purification process. 

Shiman, R., Akino, M. and Kaufman, S. Solubilization and partial purification of tyrosine hydroxylase from bovine adrenal medulla./. Biol. Chem. 246 1330-1340,1971. 

The enzyme catalyzing the formation of retinal 2 by means of central cleavage of P-carotene 1 has been known to exist in many tissues for quite some time. Only recently, however, the active protein was identified in chicken intestinal mucosa (3) following an improvement of a novel isolation and purification protocol and was cloned in Escherichia coli and BHK cells (4,5). Iron was identified as the only metal ion associated with the (overexpressed) protein in a 1 1 stoichiometry and since a chromophore is absent in the protein heme coordination and/or iron complexation by tyrosine can be excluded. The structure of the catalytic center remains to be elucidated by X-ray crystallography but from the information available it was predicted that the active site contains a mononuclear iron complex presumably consisting of histidine residues. This suggestion has been confirmed by... 

Selenoprotein A is remarkably heat stable, as seen by the loss of only 20% of activity on boiling at pH 8.0 for lOmin (Thrner and Stadtman 1973). Although selenoprotein A contains one tyrosine and no tryptophan residues, it contains six phenylalanine residues and thus has an unusual absorbance spectrum (Cone et al. 1977). Upon reduction, a unique absorption peak emerges at 238 nm, presumably due to the ionized selenol of selenocysteine, which is not present in the oxidized enzyme. The activity of selenoprotein A was initially measured as its ability to complement fractions B and C for production of acetate from glycine, in the presence of reducing equivalents (e.g., dithiothreitol). Numerous purification schemes were adopted for isolation of selenoprotein A, all of which employed the use of an anion exchange column to exploit the strongly acidic character of the protein. 

The mild acidolytic cleavage procedure noted above is used to deprotect various side chain protected peptides synthesized in solution or on chlorotrityl-resin with tyrosine O-sulfate synthons as listed in Table 4. The overall yields are significantly superior to those obtained by postsynthetic sulfation of the purified peptides, since they are typical for synthetic peptides after the final deprotection and purification step. The additional main advantage of this approach derives from a facile analytical characterization, since sulfonated byproducts at the tyrosine and tryptophan level, as well as oxidation of the methionine residues resulting from postsynthetic sulfation of tyrosine peptides are avoided. 

Most proteins have a broad characteristic absorption spectrum centered at about 280 nm. The major absorption is due to the presence of aromatic moieties in the amino acids phenylalanine, tyrosine, and tryptophan. During the a-lactalbumin purification described in this experiment, you will monitor the process by measuring the absorption at 280 nm (A2S0) of column fractions to be sure the experiment is proceeding correctly. You must recognize that you are measuring not the concentration or presence of a-lactalbumin specifically but the total amount of all proteins present. 

It has been suggested that GTF is not a chromium complex.1091 This arises from the failure to isolate a biologically active Crm complex from extracts of brewers yeast grown in a medium containing added Crm. Two fractions showed biological activity, but further purification resulted in the loss of chromium and the isolation of biologically active chromium-free compounds. One of these was largely tyramine (formed from tyrosine residues), but pure tyramine does not show GTF-type properties. The activity of this fraction must therefore be due to the minor component... 

The very short reaction times required for the alkylation of substrate 11a with benzylic bromides using Nobin as an asymmetric phase-transfer catalyst are important for the synthesis of 18F-fluorinated amino adds for use in positron-emission tomography (PET)-imaging studies. Thus, Krasikova and Belokon have developed a synthesis of 2-[18F]fluoro-L-tyrosine and 6-[18F]fluoro-L-Dopa employing a (S)-Nobin-catalyzed asymmetric alkylation of glycine derivative 11a as the key step, as shown in Scheme 8.14 [29]. The entire synthesis (induding semi-preparative HPLC purification) could be completed in 110 to 120 min, which corresponds to one half-life of18 F. Both the chemical and enantiomeric purity of the final amino acids were found to be suitable for clinical use. 

Skorey, K.I., N.A. Johnson, G. Huyer, and MJ. Gresser. 1999. A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate. Prot. Expr. Purif. 15 178-187. 

Miki Y, Morales M, Ruiz-Duenas FJ et al (2009) Escherichia coli expression and in vitro activation of a unique ligninolytic peroxidase that has a catalytic tyrosine residue. Protein Express Purif 68 208-214... 

MARQUES, I.A., BRODELRJS, P.E., Elicitor-induced L-tyrosine decarboxylase from plant cell suspension cultures I. Induction and purification. Plant Physiol., 1988,88, 47-51. 

Also N. K. Tbriks, C. D. Diltz and E. H. Fischer, Purification of the major protein-tyrosine-phosphatases of human placenta. J. Bid. Chem. 263, 6722-6730. 1988... 

Lucas, P. Lonvaud-Funel, A. (2002). Purification and partial gene sequence of the tyrosine decarboxylase of Lactobacillus brevis lOEB 9809. FEMS Microbiol. Lett., 211, 85-89. 

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