Tyrosine diazotization

Figure 4.33 Benzidine can be diazotized with sodium nitrite and HC1 for reaction with proteins through their tyrosine, histidine, or lysine side-chain groups.

Figure 19.23 The conjugation of a tyrosine-containing carrier protein and a tyrosine-containing peptide may be done using bis-diazotized tolidine to form diazo crosslinks.

Diazotization of proteins with diazonium-l/f-tetrazole in previous work has been studied in terms of reaction with histidine and tyrosine residues (50, 51). When E. coli pyrophosphatase was incubated with this reagent, there was pseudo-first-order inactivation, but analysis of the... 

Figure 151 Bis-diazotized tolidine can form cross-links with proteins through available tyrosine, histidine, or lysine residues.

Studies in solution indicate that the conformations of tyrosine-248 in the dissolved and crystalline states may be quite different. This conclusion is based on the work of Johansen and Vallee. They have examined the absorption and circular dichroic spectral properties of a carboxy-peptidase A derivative in which tyrosyl-248 is selectively labeled with the conformational probe, diazotized arsanilic acid 62, 63),... 

Zinc arsanilazocarboxypeptidase, the product of coupling crystalline carboxypeptidase with diazotized arsanilic acid, contains one arsanilazo-tyrosyl residue per molecule. No other residues are modified. Approximately 95% of the azotyrosine can be accounted for by the isolation of a peptide containing the label on tyrosine-248 (54). 

In the other method, I-labeled aniline was prepared by the chlora-mine-T method, diazotized, and allowed to react with the protein under basic conditions (Fig. 1). Presumably it couples to the phenol moiety of tyrosine residues. If this is the case, the product is analogous to that obtained by direct iodination by the chloramine-T or lactoperoxidase procedures. Papain and certain plant lectins in which cysteine residues are part of the active site were labeled without loss of activity. When variations of the chloramine-T or lactoperoxidase methods were used, 64-83% of the activity was lost. 

There are presently available several reagents, including diazotized io-dosulfanilic acid (ISA) and a Nal-lactoperoxidase (LPO) complex, that have been used to label cell membranes radioisotopically, presumably by labeling exposed tyrosine and/or histidine residues on plasma membrane proteins.Recently, the lactoperoxidase-catalyzed Nal reaction has... 

Under extreme conditions, the free amine group can be activated by diazotization and then coupled to protein tyrosine residues in a mild subsequent immobilization step. 

The Pauly test consists essentially of treating wool with diazotized sul-phanilic acid which couples with the tyrosine and histidine residues to form a brown-coloured product. These residues are found only in the cortex and, are absent from the cuticle so that the degree of staining is a measure of the breakdown of the scales. A modification of the Pauly test was recommended by Rimington J. Text. Inst., 1930, 21t, 237). The reagents required are ... 

The N-acetyl derivatives of tyrosine and histidine were purchased from Vega-Fox and diazotized by the method of Tabachnick and Sobotka (9). These reaction mixtures contained both mono- and bisdiazotized compounds which were separated by preparative TLC... 

A more selective modification, with concomitant loss of activity, has been achieved using diazotized [ °S]sulfanilic acid (130,194). The free thiol groups were protected by mercuric chloride before the tyrosine modification was performed. It was found that an incorporation of 1.5 radioactive sulfur atoms per subunit inhibited the activity. The labeled peptide was isolated and partially sequenced. By comparison with the dogfish LDH sequence the labeled tyrosine corresponds to residue 220. However, this tyrosine is nowhere near the active center of the subunit. It is most improbable that it can be associated with catalysis. 

S)-Tyrosine (264) was acylated using a Friedel-Crafts reaction providing acetyl-(iS)-tyrosine (265) as hydrochloric salt. The amino acid 265 was then transformed into hydroxy acid 266 with retention of configuration by a two step procedure consisting of diazotization of 265 and subsequent... 

A 4-(A -acetyl-L-tyrosineazo)benzamidoethyl derivative of agarose has been synthesized and used for the selective immobilization of proteins. The azo bond of the derivative can be reduced to liberate 4-aminobenzamidoethyl-agarose, which can be diazotized and recoupled to A -acetyl-L-tyrosine and subsequently to a fresh preparation of protein. Regeneration successively through five cycles is possible without detectable loss of functional groups. 

Barger (22) synthesized the base by reduction of p-hydroxyphenyl-acetonitrile with sodium. Barger and Walpole (33) later described two other syntheses. The p-hydroxy group was introduced into phenyl-ethylamine by nitration, reduction, diazotization etc., or anisaldehyde was converted to p-methoxyphenylpropionamide, from which, by Hofmann degradation and demethylation, tyramine was obtained. Hosen-mund (34) condensed anisaldehyde with nitromethane, and obtained tyramine by reduction followed by demethylation. Further syntheses of tyramine have been described by Kondo and Shinozaki (35), Slotta and Altner (36), Koessler and Hanke (37), Kindler and Peschke (38) and Buck (39, 40). A well known method of preparation is the thermal decarboxylation of tyrosine. Waser (41) obtained a 96% yield by heating the amino acid suspended in a high boiling solvent (fluorene). 

It is interesting that when tyrosine is acylated with benzoyl chloride under this condition, it forms a dibenzoyl derivative that cannot react with the diazotized sulfanilic acid whereas histidine yields only a monobenzoyl derivative that can further react with diazonium acid. However, it has been found that when pyridine is added to scavenge the generated HCl, the reaction yield might be reduced." ... 

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