Protein charge

The influence of system parameters such as protein charge, size, and concentration, ionic strength, and water content on the sizes of filled and unfilled reversed micelles has been investigated [170],... 

Grandori, R. Origin of the conformation dependence of protein charge-state distributions in electrospray ionization mass spectrometry. I. Mass Spectrom. 2003,38, 11-15. 

Characterizing the resultant complex for the amount of protein per liposome is somewhat more difficult than in other protein conjugation applications. The protein-liposome composition is highly dependent on the size of each liposomal particle, the amount of protein charged to the reaction, and the mole quantity of reactive lipid present in the bilayer construction. An approach to solving this problem is presented by Hutchinson et al. (1989). In analyzing at least 17 different protein-liposome preparations, the ratio of proteindipid content (pg protein/pg lipid) in most of the complexes ranged from a low of about 4 to as much as 675. In some instances, however, up to 6,000 molecules of a particular protein could be incorporated into each liposome. 

Isoelectric focusing also finds application in analysing the stability of biopharmaceuticals over the course of their shelf life. Repeat analysis of samples over time will detect deamidation or other degradative processes that alter protein charge characteristics. 

The effect of [Pt(NH3)s ] on the [Fe(CN)g ] oxidation of parsley PCu(I) is more difficult to interpret, but merits further comment. At pH 7.5, with [Pt(NH3)6]" (and its conjugate base) present at concentrations up to 1.31 mM, rate constants increase by a factor of 2.5 [96]. In the case of the [Co(dipic)2] oxidation (dipic is pyridine-2,6-dicarboxylate) [95], a smaller 8% increase is observed, drawing attention to the importance of size of charge. There are a number of possible explanations. These include the effect of association of [Pt(NH3)e] on the net protein charge and hence its interaction with [Fe(CN)g ] . Association of [Pt(NH3)g ] at the remote acidic patch may lead to [Fe(CN)g ] " using this site to enhance the rate. A further contributing factor may be the quite separate association of [Pt(NH3)g] and [Fe(CN)6] to give an adduct which is more redox active. 

Fig. 9.3 Flowchart illustrating how data is automatically handled and processed to yield the chemical structure of a hit. The LC/MS systems have been integrated with software to automatically deconvolute each protein charge state distribution and determine potential protein conjugation and obtain the structure of a hit by database searching.

The protein charge distribution. This is obviously an important parameter but it requires a detailed knowledge about the protein structure complemented with titration data. 

Other factors that may affect protein extraction are volume ratio of organic to aqueous phases (Vo g/V q), cosurfactant, temperature, mass transfer efficiency, protein charge, electrostatic potential of the RMs, and presence of other ions such as Ca+, Mg+, Ba+, etc. 

Rahaman and Hatton [152] developed a thermodynamic model for the prediction of the sizes of the protein filled and unfilled RMs as a function of system parameters such as ionic strength, protein charge, and size, Wq and protein concentration for both phase transfer and injection techniques. The important assumptions considered include (i) reverse micellar population is bidisperse, (ii) charge distribution is uniform, (iii) electrostatic interactions within a micelle and between a protein and micellar interface are represented by nonlinear Poisson-Boltzmann equation, (iv) the equilibrium micellar radii are assumed to be those that minimize the system free energy, and (v) water transferred between the two phases is too small to change chemical potential. 

As the ionic strength increases, the Debye screening length decreases, resulting in interparticle electrostatic interactions of shorter range. The theoretical surface potential can be further related to the net protein charge Q (Home et al, 2007) ... 

The self-assembly of caseins may be readily manipulated by processing methods that affect the integrity of native casein micelles and the character of the casein interactions in aqueous media. Examples of such procedures are (Dickinson, 2006) (i) acidification toward the isoelectric point (p/) (pH 4.6-4.8), leading to a neutralization of the net protein charge (ii) enzyme action, as exploited in the production of cheeses and fermented milks (iii) addition of divalent ions, especially, Ca2+ ions (iv) addition of sucrose or ethanol (v) temperature treatment and (vi) high-pressure treatment. 

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