Protein concentrates dimers

The second example is the SE-HPLC analysis of recombinant hGH. In this example, SE-HPLC is used for both a purity and a protein concentration method for bulk and formulated finished products. This method selectively separates both low molecular weight excipient materials and high molecular weight dimer and aggregate forms of hGH from monomeric hGH, as shown... 

Schlesinger et al. 20) concluded, on the basis of in vitro rates of dimerization, that the dimerization of enzyme subunits in vivo would not be as rapid as observed unless the subunits were compartmentalized in the cell. The in vitro rate of dimerization seemed to be based upon reoxidation and dimerization of reduced monomers and showed a maximum at 65 /ig/ml with respect to protein concentration. The in vivo process may be rather different, however, and later studies by Schlesinger and Barrett 21) with unreduced monomers would seem to change this conclusion because their rates did not have a maximum with respect to protein concentration. 

The mitogenic activities of native (tetravalent) and succinylated or acetylated (bivalent) con A were compared.336 Whereas the protein concentration-dependent, DNA synthetic response of mouse spleno-cytes to native con A exhibited a sharp peak, succinyl con A stimulated DNA synthesis over a broad range of protein concentration.336 Mixing of native con A with its dimeric, succinylated derivative at pH 4.5 resulted in the formation of hybrid molecules. A dimer species consisting of equimolar amounts of native con A protomer and its succinyl derivative was isolated, and shown3481 to have a molecular weight of50,000 at both pH 5 and pH 7. 

The vitamin D receptor acts mainly as a heterodimer with the retinoid X receptor (RXR Section 2.3.2.1). Binding of calcitriol induces a conformational change in the receptor protein, permitting dimerization with occupied or unoccupied RXR, followed by phosphorylation to activate binding to the vitamin D response element on DNA (DeLuca and Zierold, 1998). Abnormally high concentrations of 9-cis-retinoic acid result in sequestration of RXR as the homodimers, meaning that it is unavailable to form heterodimers with the vitamin D receptor (or other receptors) excessive vitamin A can therefore antagonize the nuclear actions of vitamin D (Haussler et al., 1995 Rohde et al., 1999). 

Figure 3 Equilibrium association of CAB as a function of final protein concentration ([CAB]f ). Unfolded CAB in 5 M GuHCl was rapidly diluted to a final GuHCl concentration of 2.0 M and a range of final protein concentrations. Each solution was allowed to equilibrate for three to eight hours prior to QLS analysis. The equilibrium monomer ( ), dimer ( ), and trimer ( ) concentration is shown for each final protein concentration.

The first intermediate can associate to form the dimer with an equilibrium constant,, for final protein concentrations greater than 10 /xM. The dimer equilibrium constant,, was 1.8 0.2 /xM" as calculated from the QLS data and 1.3 0.1 /xM using the HPLC data. The largest multimer, trimer, was formed from the association of a dimer and a monomer with an equilibrium constant, Kj. For this association reaction, the equilibrium constant, Kj, was 0.53 0.12 gM and 0.42 0.11 for QLS and HPLC analyses, respectively. The equilibrium constants for dimer and trimer formation are comparable using these two analytical techniques. 

Based on this equilibrium model, the association rate constant for trimer formation, kj, was 0.133 min with a half time of 5.22 minutes. Since the trimer equilibrium is rapid relative to the dimer equilibrium, the association is difficult to detect at low protein concentrations (< 33.3 /xM) using the HPLC technique since this procedure requires a total elution time of 10 minutes and results in a sample dilution of 2.5. 

Proteins exposed to even mildly denaturing conditions may partially unfold, resulting in exposure of hydrophobic residues to the aqueous solvent favoring aggregation. The aggregation process is assumed to be controlled by the initial dimerization step in a second-order reaction. Consequently, high-protein concentrations will increase the aggregation rate. 

Several lines of evidence favor the monomer as the functional form of the chemokine. Structural analysis conditions require very high concentrations of protein - levels 10- to 1000-fold greater than the protein concentrations needed for biological function, which may not occur in vivo. Also, mutational changes can be made in the primary structure that prevent multimerization and do not significantly affect function (Rajarathnam et al., 1994). Finally, the primary structure of vMIP-II, a virally encoded (human herpesvirus 8 or HHV8) chemokine, resembles that of MIP-la however, vMIP-II fails to dimerize regardless of the pH or protein concentration (personal communication from Barry L. Schweitzer). 

The monomer has a M, of 97 kDa and it is generally thought that the active form of both phosphorylase a and b is the dimer. In the case of phosphorylase b, in the absence of allosteric effectors the equilibrium lies towards the dimer at accessible protein concentrations. Phosphorylation promotes association to the tetramer by generating surface which becomes the interface of the dimer of dimers.The tetramer of phosphorylase a (and presumably phosphorylase b) is inactive and ligands have only modest effects on the association-dissociation equilibria. 

Figure 5. Time-resolved absorption changes, induced by reaction of CO2" radicals, due to intramolecular ET from the internal disulfide radical anion to Cu(ll) in the C3/C26A-N42C azurin dimer measured at 625 nm. Protein concentration was 20pAf, where T = 299K pH 7.0 0.1 Af formate 10 mM phosphate N2O saturated pulse width 1.5 ps optical path 3 cm. Time is in seconds the left panel shows the faster phase, while the right one shows the reaction taking place at the slower phase. The lower panels show residuals of the fits to the data.

Fig. 14. Quenching by peribacteroid membranes (PBMs) of the dimerization process of Fe(III) Lb in the presence of H202. Fe(III) Lb (210 /liM) was incubated with H202 (420 fj,M) for 1 h in the absence (lane 2) or in the presence of PBMs at the following protein concentrations 0.23 mg/mL (lane 3), 1.16 mg/mL (lane 4) and 2.33 mg/mL (lane 5). Lane 1 represents Fe(III) Lb alone and lane 6 and 7 PBMs plus H202 and PBMs alone, respectively (reprinted with permission from Moreau, S. Davies, M. J. Mathieu, C. Herouart, D. Puppo, A. J. Biol. Chem., 1996,271, 32557-32562).

---