Enkephalin constants

Lakowicz et al.(]7] VB) examined the intensity and anisotropy decays of the tyrosine fluorescence of oxytocin at pH 7 and 25 °C. They found that the fluorescence decay was best fit by a triple exponential having time constants of 80, 359, and 927 ps with respective amplitudes of 0.29, 0.27, and 0.43. It is difficult to compare these results with those of Ross et al,(68) because of the differences in pH (3 vs. 7) and temperature (5° vs. 25 °C). For example, whereas at pH 3 the amino terminus of oxytocin is fully protonated, at pH 7 it is partially ionized, and since the tyrosine is adjacent to the amino terminal residue, the state of ionization could affect the tyrosine emission. The anisotropy decay at 25 °C was well fit by a double exponential with rotational correlation times of 454 and 29 ps. Following the assumptions described previously for the anisotropy decay of enkephalin, the longer correlation time was ascribed to the overall rotational motion of oxytocin, and the shorter correlation time was ascribed to torsional motion of the tyrosine side chain. 

The spin-spin coupling constants [2-4] of the enkephalins in solution can be interpreted in terms of folded conformations resembling that of morphine in the placement of the residues which appear important for biological activity. X-ray crystallography and theoretical calculations (4-9) have also shown that methionine and leucine enkephalin adopt conformations similar to those concluded from NMR studies. Hence it would appear that opioid peptides can topographically resemble the opiates by assuming preferred, folded, conformations. However, earlier studies from this laboratory (TO) have shown that NMR data can be interpreted in terms of a conformationally flexible structure for methionine enkephalin. 

It is informative to compare the observed dissociation constant for the enkephalin-PS interaction with those reported for other systems. Methionine enkephalin has been shown ( ) to bind to brain membrane with a high affinity binding constant of 1.7 X IQ M and a low affinity binding constant of 1.7 x 10 ... 

Conformational analyses of JOM-13 and [L-Ala3]DPDPE have proven to be critical for the determination of the bioactive conformation of enkephalin-like peptides at the delta receptor. H-NMR studies of JOM-13 in aqueous solution revealed that this tetrapeptide exists in two distinct conformations on the NMR time scale as evidenced by two sets of resonances [63]. Large differences in the observed chemical shifts and coupling constants for the D-Cys2 residue in the two conformers suggested that the major differences between the two NMR conformers reside in the disulfide portion of the molecule however, a paucity of conformationally informative nuclear Overhauser enhancement (NOE) interactions precluded the development of a detailed structural model from the NMR studies. In order to develop such a model a thorough conformational analysis of JOM-13 was undertaken, in which the NMR data were complemented by x-ray diffraction results and by molecular mechanics calculations [64]. The results indicate that the 11-... 

Conclusively, spinal delta opioid receptors undergo a conformational change by the stimulation of spontaneously released [Met5]enkephalin and are then internalized. It is likely that the internalization of spinal delta opioid receptors becomes an irreversible process. To maintain a normal physiological function, delta opioid receptors in the plasma membrane are constantly replenished by newly synthesized delta opioid receptor protein (Fig. 3B). 

Molecularly imprinted sorbent assays represent one of the most typical applications of biomimetic use, where imprinted polymers are used as substitutes of natural antibodies in immunoassays. The assays usually involve competitive binding of an analyte with a certain quantity of labeled ligands, in which the labeled ligand unbound is proportional to the analyte added. Because dissociation constants of common imprinted polymers are around 10 6-10 9 M, competitive binding assays could easily be performed. In practice, many molecularly imprinted sorbent assays have been developed for biologically active compounds, including theophylline, diazepam [26], S-propranolol [27], morphine, Leu-enkephalin [28], cyclosporin A [29], yohimbine [30], methyl-a-glucoside [31], corticosteroid [32], atrazine [33, 34], and 2,4-D [35]. 

Methionine radicals (Met/S-Br) can oxidize tyrosine or tryptophan residues in peptides by an intramolecular process (160, 161). Mean values of distances between Tyr and Met were calculated by conformational analysis of Met-enkephalin analogs using molecular mechanicss and Monte-Carlo techniques (162). A good correlation between distances and rate constants was found. 

The micro-constants of enkephalin and tyrosine peptides... were determined according to tiie modified metiiod of complementary tri-stimulus colorimetry (CTS method) (Flaschka H, Applications of complementary tri-stimulus colorimetr). -1. Analysis of binary and ternary colorant systems, Talanta, 7, 90-106 (1960). The acid dissociation and micro-constants thus determined are summarized in Table 1 and Table 2. 

FIGURE 47.5 Representative mass spectrum of leucine-enkephalin obtained using chip with open channel emitter (a) and porous polymer monolith-assisted electrospray process from a microdevice at a constant infusion (b). (Reprinted with permission from Koerner, T. and Oleschuk, R. D Rapid Comm. Mass Spectr., 19, 3279, 2005. Copyright 2005 J. Wiley.) Conditions peptide solution 1.0 p-mol/L in 50% aqueous acetonitrile with 0.5% acetic acid, flow rate of 100 nL/min, applied voltage of 3.5 and 3.0 kV, respectively. 

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