Protein phases

Proteins with their inherent chiral nature are used in CSPs in coated and immobilized form. Bovine serum albumin was the first used in SCP in immobilized form [85] for the separation of acidic and neutral compounds. 

The ai-acid glycoprotein (AGP) was introduced as chiral selector in 1983 by Hermannson [86]. This phase found wide application in the enantiosepa-ration of basic drugs. 

---

A. Walhagen and F.-E. Edholm, Coupled-column cliromatography of immobilized protein phases for direct separation and determination of dmg enantiomers in plasma , 7. Chromatogr. 473 371-379 (1989). 

The molecular replacement method assumes similarity of the unknown structure to a known one. This is the most rapid method but requires the availability of a homologous protein s structure. The method relies on the observation that proteins which are similar in their amino acid sequence (homologous) will have very similar folding of their polypeptide chains. This method also relies on the use of Patterson functions. As the number of protein structure determinations increases rapidly, the molecular replacement method becomes extremely useful for determining protein phase angles. 

In an early, quite elaborate model for the diffusion through the stratum corneum, Michaels et al. derived an equation for diffusion through a two-dimensional brick-and-mortar structure [50], In this model, stratum corneum permeability for a given compound depended only on two parameters one was the product of the partition coefficient between the protein and the donor phase /fprot/donor and the diffusion coefficient in the protein phase >Prot the other was the product of the partition coefficient between the lipid and protein phases Aip/prot and the ratio of the diffusion coefficients in the two phases... 

Using the mineral oil-water partition coefficient to estimate the partition coefficient between the stratum corneum lipid and protein phases, and using a fixed value of Dprot = 2 x 10-7 cm2/s, Eq. 35 showed a remarkable agreement with experimental data for a value of Aip/ prot of 2 x 10-3. Unfortunately, data... 

Manipulation of mobile phase and temperature parameters can have some unusual effects on chiral separations. Variation of temperature and mobile phase composition has been reported to reverse the elution order on protein phases and polysaccharide phases (Persson and Andersson, 2001). 

The isomorphous replacement method requires attachment of heavy atoms to protein molecules in the crystal. In this method, atoms of high atomic number are attached to the protein, and the coordinates of these heavy atoms in the unit cell are determined. The X-ray diffraction pattern of both the native protein and its heavy atom derivative(s) are determined. Application of the so-called Patterson function determines the heavy atom coordinates. Following the refinement of heavy atom parameters, the calculation of protein phase angles proceeds. In the final step the electron density of the protein is calculated. 

Midostaurin (PKC- Oncology Inhibits protein Phase I/n Novartis 899... 

Once the heavy-atom position has been determined, its structure factor amplitude f h and phase an can be calculated. Since the structure factor amplitudes for the native (Fp) and derivative (Fp ) are experimentally measured quantities, it is thus possible to calculate the protein phase angle ap from the following equations ... 

Figure 6.3 Isomorphous replacement phase determination (Marker construction), (a) Single isomorphous replacement. The circle with radius Fpp represents the heavy-atom derivative, while that with radius Fp represents the native protein. Note that the circles intersect at two points causing an ambiguity in the phase angle apg and apt, represent the two possible values, (b) Double isomorphous replacement. The same construction as that in single isomorphous replacement except that an additional circle with radius Fpn2 (vector not shown for simplicity) has been added to represent a second heavy-atom derivative. Note that all three circles (in the absence of errors) intersect at one point thus eliminating the ambiguity in the protein phase angle ap. Fm and Ppy represent the heavy-atom vectors for their respective derivatives.

Korolev, S., et al. (2001). Using surface-bound rubidium ions for protein phasing. Acta Crystallogr. D 57, 1008-1012. 

Yes indicates that the peak passes the cutoff of 0.2 and should be selected as an atom (Se site) for subsequent steps in the protein phasing procedure. 

Weeks, C. M., Shah, N., Green, M. L., Miller, R. and Furey, W. (2005). Automated web- and grid-based protein phasing with BnP Acta Crystallogr. A 61 (SuppL), C152. 

Peptide Protein Phase Resin Cleavage Methyl Cleavage Ref... 

Dumetz, A.C., Chockla, A.M., Kaler, E.W., Lenhoff, AM. (2008). Effects of pH on protein-protein interactions and implications for protein phase behaviour. Biochimica et Biophysica Acta, 1784, 600-610. 

Ahamed, T., Ottens, M., van Dedem, G.W.K., van der Widen, L.A.M. (2005). Design of self-interaction chromatography as an analytical tool for predicting protein phase behaviour. Journal of Chromatography A, 1089, 111-124... 

Figure 7.8 Con Focal microscopy images for some freshly prepared emulsions containing 0.14 wt% locust bean gum, made with either skim milk powder (a, b) or sodium caseinate (c, d), and with no added K-carrageenan (a, c) or 0.02 wt% K-carrageenan (b, d). As the protein phase was fluores-cently stained, it appears w hite. Scale bars correspond to 40 pm. Reproduced from Vega el al. (2005) with permission.

Pugnaloni, L.A., Ettelaie, R., Dickinson, E. (2003b). Do mixtures of proteins phase separate at interfaces Langmuir, 19, 1923-1926. 

Milk fat plays a very important role in the development of texture in cheese. Reduced-fat cheeses tend to be firmer and more elastic than cheeses with a higher fat content. Undoubtedly the presence of a more dense protein matrix results in a firmer cheese. The precise role of fat in cheese texture is not well understood, since problems of increased firmness can be partially overcome by increasing the MNFS. Studies by Green et al (1981) on the texture of cheeses made from concentrated milk suggest a possible role of fat in cheese firmness. Reduced fat in the curd would result in a smaller fat-protein interfacial area and an increased separation between fat globules. The capacity of the fat and protein phases of cheese to move in relation to each other would be reduced and would consequently result in a firmer cheese. 

An experimental complication is the difficulty in effecting molecular interaction between the components. The usual technique for preparing lipid-protein phases in an aqueous environment is to use components of opposite charge. This in turn means that the lipid should be added to the protein in order to obtain a homogeneous complex since a complex separates when a certain critical hydrophobicity is reached. If the precipitate is prepared in the opposite way, the composition of the complex can vary since initially the protein molecule can take up as many lipid molecules as its net charge, and this number can decrease successively with reduction in available lipid molecules. It is thus not possible to prepare lipid— protein—water mixtures, as in the case of other ternary systems, and to wait for equilibrium. Systems were prepared that consisted of lecithin-cardiolipin (L/CL) mixtures with (a) a hydrophobic protein, insulin, and with (b) a protein with high water solubility, bovine serum albumin (BSA). 

Observations were made of lipid-protein phases in which the structure is determined mainly by the protein. Raman spectroscopy is a useful method for structure analysis of such phases. The structures described above were analyzed successfully by an x-ray diffraction technique. Lipid-protein complexes, however, are often amorphous, and alternative methods to study their structures are therefore needed. It was demonstrated that Raman spectroscopy can be used to obtain structural information about lipid-protein interaction (16, 17). It is thus possible to determine the conformation as well as the type of environment of the lipid molecules. With the protein, interpretation is more complicated. It is usually possible to determine whether the complex has the same protein conformation as the component used in the preparation, or, if a change occurs, it may be possible to correlate it with denaturation of the pure protein. For complexes formed by long-chain alkyl phosphates and insu-... 

The protein phases were the first to be developed and are still in use for the chiral resolution of different racemates. Because both BSA and HSA are similar in structure, they have similar enantiorecognition capabilities. Warfarin and... 

The optimization of chiral resolution on protein phases by flow rate is seldom carried out. However, Kirkland et al. [60] optimized the chiral resolution of... 

---