Protein critical values

This proposal describes the development of a new, systematic approach for qualitatively and quantitatively studying surface-biomolecule interactions by matrix-assisted laser desorption ionization (MALDl) mass spectrometry (MS). This methodology is being developed because of the profound importance that surface-biomolecule interactions play in applications where biomaterials come into contact with complex biological fluids, it can readily be shown that undesired reactions occurring in response to surface-biomolecule contact (protein adsorption, biofouling, immune response activation, etc.) lead to enormous economic and human costs. Thus, the development of analytical methodologies that allow for efficient assessment of the properties of new biomaterials and/or the study of detailed fundamental processes initiated upon surface-biomolecule contact are of critical value ... 

As an example, we will consider the molecular dynamical behavior of egg white lysozyme. The temperature dependence of mobility of fluorescence, spin and Mossbauer labels attached to lysozyme was found to be similar to other investigated proteins the monotonic increase typical for rigid polymers in dry states and in samples with water content (wt) was less than the critical value (wtcr) and drastically burst when wt > wtcr at T > 200 K took place (Frolov et al., 1978 Likhtenshtein, 1979). At similar conditions, experiments on the temperature dependence of heat capacity indicated only a monotonic steady increase for rigid organic material. Recently, in the fully dried lysozyme crystal, similar monotonic behavior of heat capacity was observed in temperatures between 8 and 30°C. At D20 content more than 24 wt %, a slight deviation from the monotony was observed at temperatures above approximately 185 K, which most probably is due to the eutectic melting of NaCl/2H20 present in the samples to prevent water crystallization (Miyazaki et al., 2000). 

Crystal nucleation rates, expressed as the number of nuclei formed per unit volume per unit time, increase with protein solubility. Higher solubility leads to increased molecular encounters in solution and reduced levels of supersaturation required for spontaneous nucleation. Nucleation rates typically show a high-power dependence on protein supersaturation, and so empirically increase rapidly above a critical value... 

When a protein molecule adsorbs, an area of interface of the order of 100 A has to be cleared for adsorption to occur (Section III,B). It seems reasonable to assume that once an adsorbed molecule has been compressed until its area in the interface, due to pressure displacement of segments, falls below this critical value, it will be unstable in the adsorbed state and will desorb. This transition state for desorption may be reached in two ways (1) at constant interfacial pressure and total area, by fluctuations in energy of the adsorbed molecules about the mean value, resulting in certain molecules achieving the transition state configuration (2) by compression of the film, thus increasing the interfacial pressure and decreasing the molecular area until the latter has been reduced to the critical value. 

When protein solutions are shaken, insoluble protein is often seen to separate out (Bull and Neurath, 1937). The coagulation occurs at the interface and may be observed when protein is allowed to adsorb from solution at a quiescent interface (Cumper and Alexander, 1950) or when spread protein monolayers are compressed (Kaplan and Frazer, 1953). This is an interesting type of phase separation in which a three-dimensional coagulum is formed from the two-dimensional monolayer, once a certain critical value of the interfacial pressure is exceeded. The concentration of protein in the monolayer when the critical pressure is reached may be thought of as the solubility in the interface under those conditions. When this concentration is exceeded, precipitation occurs. A simple model may help to illustrate how free energy considerations govern the coagulation. 

For the extraction of proteins, aqueous two-phase systems (ATPS) are preferred over organic solvents, which usually denature the proteins and render them biologically inactive. They consist of polyethylene glycol (PEG), and a salt (e.g., potassium phosphate) or dextran in water. At concentrations above a critical value, the mixture separates into two phases—one rich in PEG and the other in dextran or salt. In industrial systems, salts are more commonly used because they are relatively inexpensive as compared to dextran. The MW, charge and surface properties of the protein decide how the protein partitions in the system. The nature of the phase components, the MW of the polymer, and the concentration and type of salt used also affect the distribution. ... 

This was successfully applied to the case of globular proteins, using a value of 1 or 1.2 for the exponent a and changing a for the protein critical charge (Zpr) at the onset of the complexation (Figure 7). Given the proportionality between k and the ionic strength /jl, this relationship was written... 

When a protein solution is heated, a gel may be formed. This occurs with well-soluble globular proteins, if the protein concentration is above a critical value c0. The gel is only formed after at least part of the protein has been heat denatured—e.g., as inferred from a change in the DSC curve—and the gel formation is irreversible upon cooling see Figure 17.14d. Gel formation is a relatively slow process, taking at least several minutes and possibly hours. 

Data are presented in several forms for many of the partitioned materials. The concentration of material in the salt phase and the partition coefficient (concentration in the PEG phase / concentration in the salt phase = K) are plotted as functions of salt concentration and pH on semi-log scale. On the log axis, 1E0 represents 1 x 10(0) or 1 2E3 represents 2 x 10(3) or 2,000. A K value of 1 indicates no preference for either phase the concentration is the same in both. It is this value of unity that is critical. Values greater than 1 indicate a preference for the top (PEG) phase and values less than 1 indicate the bottom (salt) phase. In several cases, precipitation of protein occurs at the... 

These results are of general significance for the study of biological rhythms as they show how the continuous variation of certain control parameters can lead to the emergence of a rhythm in the course of development of an organism. Here, the level of certain proteins augments once the amoebae begin to synthesize the components of their intercellular communication system after starvation. As soon as the concentration of the cAMP receptor and the activity of enzymes such as adenylate cyclase and phosphodiesterase reach a critical value, oscillations appear spontaneously. Rinzel Baer (1988) have shown, however, that a certain delay separates the time at which the parameters cross their bifurcation values and the moment at which oscillations... 

The first parameter considered is Vj, which measures the maximum rate at which PER is degraded. When all other parameter values remain as in fig. 11.7, numerical simulations show that sustained oscillations occur in a window bounded by two critical values of this parameter, close to 0.45 and 2.6, respectively (in p-M/h if a scale of xM is chosen for concentrations). In this interval of values, the period of the oscillations increases from a value close to 19.7 h up to a value close to 64 h. The period range in the window of values depends on other parameters. Thus, for a larger value of the rate of protein synthesis measured by ks, the period varies as a function of from 15.9 to 62.1 h (fig. 11.9). 

A much more complex behaviour is observed for the process of penetration of various proteins into phospholipid monolayers. This behaviour depends strongly on the protein and the solution properties although some common features are observed. Fainerman et al. [116] studied the P-lactoglobulin penetration dynamics into DPPC monolayers. For a (i-lactoglobulin bulk concentration of 510 mol/l and molar areas of the lipid larger than the critical value, A > A, first order phase transitions are observed. Thus, two-dimensional condensed phase are formed although at these molar area values the pure DPPC monolayer exists only in the fluid-like state and does not form any domains. The first-order phase transition in the DPPC monolayer becomes visible by the characteristic break point in the dynamic surface pressure curve Fl(t) (see Fig. 4.50). 

The solvoit slope bsis equal to 10% for both proteins. This value in the same range of that (83-9.2%) finind for Tip residue in pure solvent (Weber et al. 1984, Scarlata et a1. 1984 Rholam el al.1984.) The first critical temperature observed was found at -30°C and for the sialylatcd and asialylated forms, respectively At this temperature, the amphiude (a) and the angle (0) of the rotatirai of the buried 1 ip residues calculated fiom EqnsS.19andS.20 ... 

A second break in the Y-plot occurs at a critical temperature equal to -9 C, instead of - 16 C observed for the asialylated form. At -9 C (in the sialylated protein), the values of a and 0 corresponding to the amplitude and the angle of rotation of the Trp residue of the protein surface of the sialylated protein were 0.077 and 13° of arc. These values are close to those (0.084 and 14° of arc) obtained for the asialylated protein. 

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