Desorption denatured protein

Process 3 The desorption rate is much slower, particularly after long contact times. They assume that fully denatured protein does not desorb. 

Desorption of Denatured Protein. The energetics of protein binding to hydrophobic surfaces probably involves the unfolding of protein to achieve optimal hydrophobic bonding, but is undoubtedly entropically driven. In our... 

The question remains whether the denatured protein molecule regains its native structure on desorption. Soderquist and Walton extensively studied the structure of three plasma proteins—albumin, y-globulin and fibrinogen—on desorption from different co-polypeptide (amino acid copolymers) and silicone surfaces under different pH and ionic strength conditions [88]. Circular dichroism (CD) analysis of the surface desorbed proteins showed a marked decrease in the a-helical content for albiunin and fibrinogen, whereas y-globulin lost most of its P-sheet structure. The reduction... 

In addition to chemical reactions, the isokinetic relationship can be applied to various physical processes accompanied by enthalpy change. Correlations of this kind were found between enthalpies and entropies of solution (20, 83-92), vaporization (86, 91), sublimation (93, 94), desorption (95), and diffusion (96, 97) and between the two parameters characterizing the temperature dependence of thermochromic transitions (98). A kind of isokinetic relationship was claimed even for enthalpy and entropy of pure substances when relative values referred to those at 298° K are used (99). Enthalpies and entropies of intermolecular interaction were correlated for solutions, pure liquids, and crystals (6). Quite generally, for any temperature-dependent physical quantity, the activation parameters can be computed in a formal way, and correlations between them have been observed for dielectric absorption (100) and resistance of semiconductors (101-105) or fluidity (40, 106). On the other hand, the isokinetic relationship seems to hold in reactions of widely different kinds, starting from elementary processes in the gas phase (107) and including recombination reactions in the solid phase (108), polymerization reactions (109), and inorganic complex formation (110-112), up to such biochemical reactions as denaturation of proteins (113) and even such biological processes as hemolysis of erythrocytes (114). 

One of the most important causes of poor resolution in protein and large peptide analysis with conventional phases is slowness of mass transport and of sorption/desorption. When selecting a column for a protein separation, it is important to look for obtaining rapid separations in order to limit protein denaturation. 

Immunoaffinity chromatography is probably the most highly specific of all forms of bioaffinity chromatography. However this technique has some disadvantages such as this technique relatively high cost, leakage of ligands may accur from the column and sometimes the desorption procedure results in partial denaturation of the bound protein [24]. 

Fig. 13. Most of the kinetic models which might be applicable to protein adsorption (see Refs.70 73)) k is rate constant, subscript a and d are adsorption and desorption respectively, 1 and 2 are adsorption states — usually native and denatured...

Beissinger s and Leonard s model (Fig. 13 d) accounts for desorption of both native and denatured adsorbed species (States 1 and 2, respectively). They used the classical Langmuir-Hinshelwood model for catalytic reactions (the surface is the catalyst for conformational change or denaturation of the adsorbed protein), which assumes equilibrium at a steady state between adsorbed and solution molecules. They show ... 

Fig. 24. Matsuda, et al. s model of the protein adsorption/denaturation/aggregation/desorption/ delamination process involved in the blood interactions of materials (from Ref. 127 , p. 357)...

Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS) Mass spectra of native and denatured antibodies were obtained with a PerSeptive Biosystems (Farmingham, MA.) Voyager Elite mass spectrometer operated in the linear mode with a Laser Sciences Inc., 337 nm nitrogen laser. hAB-1 was denatured by boiling the sample in 1.0 M guanidine-HCl, 50 mM Tris pH 7.5 buffer. Native and denatured samples were diluted with 20 mM Tris, 10 mM octylglucoside (Tris/OG) pH 6.8 buffer prior to MALDl-MS analysis. Proteins were spotted on the sample plate as a sandwich between two layers of the matrix. The bottom layer consisted of 100 mM sinapinic acid in acetonitrile and the top layer consisted of 50 mM sinapinic acid in 30% acetonitrile / 70% H2O / 0.07% TFA. The m/z scale of the instrument was calibrated using a Hewlett-Packard protein standard mixture. 

The VERSE method was extended to describe the consequences of protein de-naturation on breakthrough curves in frontal analysis and on elution band profiles in nonlinear isocratic and gradient elution chromatography [45]. Its authors assumed that a unimolecular and irreversible reaction taking place in the adsorbed phase accormts properly for the denaturation and that the rate of adsorption/desorption is relatively small compared with the rates of the mass transfer kinetics and of the reaction. Thus, the assumption of local equilibrium is no longer valid. Consequently, the solid phase concentration must then be related to the adsorption and the desorption rates, via a kinetic equation. A second-order kinetics very similar to the one in Eq. 15.42 is used. 

The size of a molecule is an important feature because proteins form multiple contacts with the surface (e.g., 77 contact points in the case of the albumin molecule and 703 contact points in the case of the fibrinogen molecule adsorbed on silica [10]). Multipoint binding usually causes adsorption irreversibility having a dynamic nature in the absence of irreversible denaturation. The rates of desorption are, as a rule, much lower than those of adsorption, and in many cases it is virtually impossible to attain the equilibrium state desorbing the adsorbed protein [11]. In other words, the formation of one or several bonds with the surface increases the probability of adsorption of neighboring sites of the same molecule. On the other hand, the desorption of a protein molecule requires the simultaneous rupture of a large number of bonds and, for kinetic reasons, equilibrium is not attained [12-14], This corresponds to a considerable difference between the activation energies for the adsorption and desorption processes [15,16],... 

The presence of the solid surface imposes new conditions onto the disposition of reactants, compared with the homogeneous case (section C2.14.4.T). Adsorption is often observed to approach a plateau, yet is irreversible with respect to dilution. The plateau must therefore arise because no more space is available for adsorption, rather than through a dynamic adsorption-desorption equilibrium and it can be inferred that the dissolved biopolymer does not adsorb to its preadsorbed congeners. This behaviour is by no means universal it has been proposed that the plaques associated with spongiform encephalopathies arise through the native, normally soluble Pr protein being partially denatured upon contact with a surface to become the pathogenic PrP form, to which the PrP can adhere to form multilayers. 

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