Protein denaturation with urea

Total nitrogen content, X, of both untreated and deproteinized mbbers, is shown in Table 1. The total nitrogen content of HANR was reduced to 0.017 wt% after enzymatic deproteinization (E-DPNR), as reported in the previous study (Tanaka et al., 1997). On the other hand, it was reduced to 0.020 wt% after the treatment with urea, being similar to the nitrogen content of E-DPNR. This implies that most proteins present in NR are attached to the mbber with weak attractive forces. To remove further the proteins, the treatment with nrea was carried out after the enzymatic deproteinization of HANR latex. The nitrogen content of the resulting rubber, EU-DPNR, was 0.008 wt%, less than that of E-DPNR and U-DPNR. This suggests that the most of proteins are removed by denaturation with urea, whereas the residue must be removed with proteolytic enzyme in conjunction with nrea. 

The ESR spectrum of the enzyme denatured with urea is remarkably different from that obtained after prolonged treatment of the spin labeled enzyme with H2O2, although both reagents unfold the protein architecture. 

It appears that ths method is quite specific and that no serious interference need be expected from other than sulfhydryl-containing substances. It should be pointed out, however, that substances which form stable complexes or insoluble salts with silver ions (e. g., cyanide, iodide, and sulfide) may be expected to interfere. Possible steric impediments to the access of the Ag(NHs) ion to the —SH groups should be kept in mind. The process involved is essentially one of mercaptide formation and the limitations of such a process (particularly with respect to the specificity of the diver mercaptide formation) diould also be kept in mind. The procedure has been applied to the determination of —SH grmips in proteins denatured with guanidine hydrochloride and with urea (24,123). It has been shown that the addition of an excess of standard potasdum p-chloro-mercuribenzoate prior to the titration with silver nitrate effectively blocks... 

O. D. Monera, C. M. Kay, and R. S. Hodges, Protein Sci., 3,1984 (1994). Protein Denaturation with Guanidine Hydrochloride or Urea Provides a Different Estimate of Stability Depending on the Contributions of Electrostatic Interactions. 

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

Table V shows the results of this analysis for the Pn-helix fraction of several proteins denatured by heat, cold, acid, and Gdm HCl/urea. There is rather good consistency among the estimated Pn-helix contents for proteins denatured by a given agent, except for acid-denatured proteins, which show more variability. The chemically denatured proteins have 30 5% Pn-helix content near 0°C. At the other extreme, heat-denatured proteins have Pn-helix contents near 0%, with lysozyme having the highest value (8%). Although there are only two examples of cold-denatured proteins in Table V,2 they both have Pn-helix contents of about 20%. Acid-denatured proteins have Pn-helix contents ranging from 0 to 16%.

Figure 5. Proton decoupled 19F-NMR spectrum of pABG5 / -glucosidase inactivated with 2F/ ManF (conditions as described in text). Spectra were recorded on a 270 MHz Bruker/Nicolet instrument using gated proton decoupling (decoupler on during acquisition only) and a 90° pulse angle with a repetition delay of 2s. A spectral width of 40,000 Hz was employed and signal accumulated over 10,000 transients for the native protein and 30,000 transients for the denatured protein in 8M urea, (a) Full spectrum with expansion below it (b) Expansion of spectrum of denatured/dialyzed enzyme.

Salt (ionic strength) gradients in lEC discussed in Section 5.4.3.3 are frequently used in the separation of complex peptides, proteins, and other biopolymer samples as a complementary technique to RP solvent gradient separations, often in a 2D setup [99,100]. The gradients usually start at a low salt (chloride, sulfate, etc.) concentration and typically run from 0.005 to 0.5 M. A buffer is used to control the pH acetonitrile and methanol may be added to improve the resolution and urea to improve the solubility of proteins that are difficult to dissolve. Ion exchangers with not strongly hydrophobic matrices usually prevent protein denaturation in aqueous mobile phases. 

Requirements for Protein Translocation across a Membrane The secreted bacterial protein OmpA has a precursor, ProOmpA, which has the amino-terminal signal sequence required for secretion. If purified ProOmpA is denatured with 8 M urea and the urea is then removed (such as... 

It is known that heat-denaturated proteins have higher ellipticity than the proteins denatured by guanidinium chloride or urea. This is usually explained by the assumption that they have some regular residual structure (Tanford, 1962,1968). At the same time, all attempts to measure the heat effect associated with disruption of this residual structure by guanidinium chloride or urea have failed (Pfeil and Privalov, 1976b Pfeil, 1981,... 

---