Tyrosine residues ribonuclease

By comparing time-resolved and steady-state fluorescence parameters, Ross et alm> have shown that in oxytocin, a lactation and uterine contraction hormone in mammals, the internal disulfide bridge quenches the fluorescence of the single tyrosine by a static mechanism. The quenching complex was attributed to an interaction between one C — tyrosine rotamer and the disulfide bond. Swadesh et al.(()<>> have studied the dithiothreitol quenching of the six tyrosine residues in ribonuclease A. They carefully examined the steady-state criteria that are useful for distinguishing pure static from pure dynamic quenching by consideration of the Smoluchowski equation(70) for the diffusion-controlled bimolecular rate constant k0,... 

Ribonuclease is an enzyme with 124 amino acids. Its function is to cleave ribonucleic acid (RNA) into small fragments. A solution containing pure protein, with no other ions present except H+ and OH- derived from the protein and water, is said to be isoionic. From this point near pH 9.6 in the graph, the protein can be titrated with acid or base. Of the 124 amino acids, 16 can be protonated by acid and 20 can lose protons to added base. From the shape of the titration curve, it is possible to deduce the approximate pATa for each titratable group.1-2 This information provides insight into the environment of that amino acid in the protein. In ribonuclease, three tyrosine residues have "normal values of pATa(=10) (Table 10-1) and three others have pA a >12. The interpretation is that three tyrosine groups are accessible to OH, and three are buried inside the protein where they cannot be easily titrated. The solid line in the illustration is calculated from pA"a values for all titratable groups. 

Gobrinoff, M. J. 1967. Exposusre of tyrosine residues in proteins. Reaction of cyanuric fluoride with ribonuclease, a-lactalbumin, and /3-lactoglobulin. Biochemistry 66, 1606-1614. 

Ribonuclease contains no tryptophan. The absorption near 280 nm is almost entirely resulting from the 6 tyrosine residues. The ionization of tyrosine produces a marked shift to longer wavelengths in the absorption spectrum. The ionization can be monitored near 295 nm. Shugar (293) was the first to point out the abnormal behavior of 3 of the tyrosine residues on alkaline titration. Three titrate normally with apparent pK values near 10, but three do not titrate until much more alkaline pH values have been reached and irreversible alkaline de-naturation has set in. Some typical spectra and difference spectra are... 

Also, HPLC methods with electrochemical or fluorescent detection are used (H19, M3). In proteins, dityrosine can be estimated by immunochemical methods employing dityrosine-specific antibodies (K5). Measurements of o,o -dityrosine and o-tyrosine levels in rat urine express dityrosine contents in skeletal muscle proteins, and have been proposed as the noninvasive oxidative stress test in vivo. One should be aware, however, that A-formylkynurenine, also formed in protein oxidation, has similar fluorescence properties as dityrosine (excitation 325 nm, emission at 400-450 nm) (G29). Also, oxidation of mellitin when excited at 325 nm produces an increase in fluorescence at 400—450 nm, despite the fact that mellitin does not contain tyrosine. Oxidation of noncontaining Trp residues ribonuclease A and bovine pancreatic trypsin inhibitor with "OH produces loss of tyrosine residues with no increase in fluorescence at 410 nm (S51). There are also methods measuring the increased hydrophobicity of oxidized proteins. Assays are carried out measuring protein binding of a fluorescent probe, 8-anilino-l-naphthalene-sulfonic acid (ANS). Increase in probe binding reflects increased surface hydrophobicity (C7). 

The tyrosines of bovine pancreatic ribonuclease (RNase) appear to be a case in point. Three of the total of six tyrosine residues per RNase molecule titrate reversibly with a normal pK of about 10, but the other three titrate only at much higher pH and then irreversibly (Shugar, 1952 Tanford et al., 1955a). These results suggest that the RNase molecule has to undergo a profound structural rearrangement before the three anomalous tyrosines become accessible to titration. Furthermore, the absorption of RNase due to tyrosine residues at about 280 m/i exhibits a hyperchromic effect presumably as a result of the special environment of the three anomalous tyrosines. 

Sage and Singer (1958, 1962) showed that ribonuclease could not only be recovered from neutral ethylene glycol into aqueous solution with essentially full retention of enzymatic activity, but that this was so even after all six of its tyrosine residues had been converted to the phenoxide ion form in ethylene glycol. This is in contrast to the situation in water solutions of this protein, in which the titration of more than three of the six tyrosines results in an essentially instantaneous irreversible loss of enzymatic activity (Sela and Anfinsen, 1957). This suggests the interesting possibility that the irreversible transition that occurs in aqueous solutions... 

A spectrophotometric titration of the phenolic groups of myosin and its subunits has been reported by Stracher (1960). The data resemble those shown for ribonuclease in Fig. 11. About two-thirds of the tyrosine residues are titrated normally, and about one-third appear inaccessible in native myosin. An interesting feature is that 6 M urea has no effect at all on the titration curve. 

Poly-L-tyrosine absorbs strongly at 1515cm . A band at this frequency has been found in a number of proteins (e.g., a-keratin, ribonuclease, and insulin) and was considered to be due to the tyrosine residue (Bendit, 1967). This assignment was confirmed by examination of the spectra of deuterated proteins which usually exhibit this band at 1513 cm . ... 

Noronha M, Lima JC, Paci E et al (2007) Tracking local conformational changes of ribonuclease A using picosecond time-resolved fluorescence of the six tyrosine residues. Biophys J 92 4401 1414... 

Luse and M(iLaren (1963) have reviewed published research on the photolysis products and quantum yields tor the destruction of amino acids and have attributed the photochemical inactivation of the enzymes chymo-trypsin, lysozyme, ribonuclease, and trypsin by UV light at 254 m i primarily to destruction of the cystyl and tryptophyl residues. The destruction of these residues in proteins was suggested to be a function of the product of the number of residues present, the molecular extinction coefficient, and the quantum yield for destruction of each residue. Cysteine and tryptamine were identified among the irradiation products from cystine and tryptophan, respectively. Tyrosine, histidine, and phenylalanine were also shown to be degraded by UV, histidine yielding histamine, urocanic acid, and other imidazole derivatives, and phenylalanine yielding tyrosine and dihydroxyphenylalanine. Destruction of these three amino acids was not considered to contribute appreciably to the enzyme inactivation. 

Phe) is dominated by the tryptophan absorbance, whereas ribonuclease (RNase 0 Trp, 6 Tyr, 3 Phe), which (unusually) contains no tryptophan residues, is more characteristic of the tyrosine side chains... 

In contrast to the case of tryptophan the photoreactions with tyrosine and histidine probably involve hydrogen atom transfer as the primary step. There are several indications for this. First, 0-methylated tyrosine (p-methoxy phenylalanine) did not show any photo-CIDNP effect and its reactivity as a photo-reductant towards flavins is strongly reduced (19). Similarly, 1-N-methyl histidine is not polarized at high pH (> 7.5), when no abstractable hydrogen is present. Secondly, in the protein ribonuclease A, which has a well known 3-dimensional structure, the residues Tyr 92 and His 105 have exposed rings, but their OH and NH protons are hydrogen bonded to backbone carbonyl groups. 

Tryptophan (Trp), tyrosine (Tyr), cystine (Cys), and phenylalanine (Phe) moieties play a determinant role regarding UV light-induced chemical alterations in many proteins. After the absorption of light by these moieties, in most cases mainly by Trp and Tyr, they undergo photoionization and participate in energy-and electron-transfer processes. This not only holds for structural proteins such as keratin and fibroin [11], but also for enzymes in aqueous media such as lysozyme, trypsin, papain, ribonuclease A, and insulin [7]. The photoionization of Trp and/or Tyr residues is the major initial photochemical event, which results in inactivation in the case of enzymes. A typical mechanism pertaining to Trp residues (see Scheme 8.3) commences with the absorption of a photon and the subsequent release of an electron. In aqueous media, the latter is rapidly solvated. By the release of a proton, the tryptophan cation radical Trp is converted to the tryptophan radical Trp. ... 

Signals in the aromatic region of the n.m.r. spectrum of colipase have been assigned to L-histidine residues. L-Histidine C-2 proton signals have been used in studies of the binding to ribonuclease A of H, edta and of 2 -deoxy-2 -fluorouridilyl-(3, 5 )-adenosine. L-Phenylalanine, L-tyrosine, and methionine side-chain proton resonances were used as monitors for the unfolding and stabilization of ribonuclease." ... 

Padros E, Morrows A, Manosa J, Dunach M 1982 The state of tyrosine and phenylalanine residues in proteins analyzed by fourth-derivative spectrophotometry Histone HI and Ribonuclease A. Eur J Biochem 127 117-122... 

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