Guanidine hydrochloride polypeptides

The limited solubility of membrane proteins and related polypeptides in aqueous mobile phases can also cause problems. These could be solved, e.g., by adding guanidine hydrochloride (6 M) or urea (8 M) to the portion of initial eluent used for sample preparation 69). The urea was always eluted in the breakthrough volume of the column. Thus, the retained hydrophobic polypeptides might have been temporarily precipitated upon the column. Collagen chains, dissolved in 0.5 M acetic acid, were successfully separated by RP-HPLC through gradients of 0.1 M TFA/acetonitrile 70> or (0.05 M ammonium bicarbonate + TFA)/ tetrahydrofuran 57>. 

Additional size estimates of the 5 M guanidine hydrochloride subunit can be made from the Svedberg equation (18), using the s°0 w and D 0.w values cited in Section II,F, and from the empirical equations of Tan-ford and associates (32, 33), which relate s and [17] values in concentrated guanidine hydrochloride solvents to polypeptide chain length. These calculations yield molecular weights of 21,300, 20,700, and 19,800, respectively, for the E. coli pyrophosphatase subunit (13). 

As shown in the preceding section, in the presence of 5 M guanidine hydrochloride the six subunits of the enzyme were separated from one another and opened up to form randomly coiled, single polypeptide chains. These were catalytically inactive (13). When the guanidine hydrochloride was removed by dialysis, enzymic activity was restored, provided the protein was in a reducing environment during the dialysis (Table II). Under optimal conditions (No. 5 of Table II) there was 80-90% recovery of activity. If there was opportunity for persistent disulfide... 

More recently Lapanje and Tanford (59) have reported osmotic pressure measurements for reduced protein polypeptide chains in 6M guanidine hydrochloride. Second virial coefficient data and intrinsic viscosity data are combined by these authors to yield unperturbed dimensions of randomly coiled proteins. The result is assentially identical with that obtained earlier from viscosity data alone. 

It is apparent that while incubation in solutions of low guanidine hydrochloride concentration may sometimes cause inactivation (carbonic anhydrase), it may also promote pro r refolding (chymotrypsinogen). In addition, replacement of a strong denaturant with a weaker one is an approach which can be used to successfully refold polypeptide chains. These data suggest that the rate of removal of the denaturant agent can influence yield of active protein. In this respect, time is an important refolding parameter. 

The bovine enzyme was demonstrated to be composed of polypeptide chains that are identical and not covalently linked from the following studies. Alanine is the sole NHa-terminal amino acid and threonine is the sole COOH-terminal amino acid of the enzyme (127,128). Physicochemical studies performed on enzyme completely denatured by high concentrations of guanidine hydrochloride (102,129,130) indicated subunits of identical size. 

Additional steps are necessary if the initial protein sample is actually several polypeptide chains. SDS-gel electrophoresis under reducing conditions should display the number of chains. Alternatively, the number of distinct N-terminal amino acids could be determined. For a protein made up of two or more polypeptide chains held together by noncovalent bonds, denaturing agents, such as urea or guanidine hydrochloride, are used to dissociate... 

Tanford (30), which pertains to the unperturbed end-to-end distance of a polypeptide chain gives a value of 59 +7A for a polypeptide of 50 residues, which is quite close to the above result. However, the latter is not the unperturbed distance (since guanidine hydrochloride is not a 0 solvent) and the actual distance would be expected to be larger than the unperturbed distance. It should be kept in mind that in the reduced state, transfer efficiency is very low and the uncertainty in the distances distribution function is increased. Hence, this result should be discussed with caution. 

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