Pi, value

Epiactis prolifera. Extracts of this relatively common species contain two toxins, epiactolysins A and B (13). They have the same molecular weight, namely about 19,500, but differ in pi values as determined by isoelectric focusing. Both lack... 

However, as stated above, the partition coefficients measured by the shake-flask method or by potenhometric titration can be influenced by the potenhal difference between the two phases, and are therefore apparent values which depend on the experimental condihons (phase volume ratio, nature and concentrahons of all ions in the solutions). In particular, it has been shown that the difference between the apparent and the standard log Pi depends on the phase volume raho and that this relationship itself depends on the lipophilicity of the ion [80]. In theory, the most relevant case for in vivo extrapolation is when V /V 1 as it corresponds to the phase ratio encountered by a drug as it distributes within the body. The measurement of apparent log Pi values does not allow to differentiate between ion-pairing effect and partihoning of the ions due to the Galvani potential difference, and it has been shown that the apparent lipophilicity of a number of quaternary ion drugs is not due to ion-pair partitioning as inihally thought [80]. 

A form of this approach has long been followed by RT Corporation in the USA. In their certification of soils, sediments and waste materials they give a certified value, a normal confidence interval and a prediction interval . A rigorous statistical process is employed, based on that first described by Kadafar (1982,), to produce the two intervals the prediction interval (PI) and the confidence interval (Cl). The prediction interval is a wider range than the confidence interval. The analyst should expect results to fall 19 times out of 20 into the prediction interval. In real-world QC procedures, the PI value is of value where Shewhart (1931) charts are used and batch, daily, or weekly QC values are recorded see Section 4.1. Provided the recorded value falls inside the PI 95 % of the time, the method can be considered to be in control. So occasional abnormal results, where the accumulated uncertainty of the analytical procedure cause an outher value, need no longer cause concern. 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

The immobilization procedure performed in two stages allows one to exclude the detrimental effect of acid on the entrapped proteins [44,71,86]. It was demonstrated [87] by the example of a set of oxidases that their activity was retained if the entrapment was carried out at a pH as close to their isoelectric point (pi) as possible. Because the pi value of most enzymes is in the neutral region, the two-stage procedure favors the retention of their functionality. Therefore, the pH shift to the optimal region provides a means of extending the sol-gel entrapment to a wide range of enzymes [45,71]. 

RNase A is a basic protein with an isoelectric point (pi) of 9.45 and a net positive charge in neutral solution.35 However, the conversion of positively charged lysine side chains to polar, but neutrally charged, methylol adducts would be expected to lower the pi of formalin-treated RNase A. To explore this further, RNase A was treated with 5% formalin and analyzed by isoelectric focusing (IEF) gel electrophoresis. Figure 15.5a shows that the pi values were shifted into the pH 6.0-7.4 range. Figure 15.5b shows the results of IEF... 

This technique is also utilized in the biopharmaceutical industry to determine product homogeneity. Homogeneity is best indicated by the appearance in the gel of a single protein band, exhibiting the predicted pi value. Interpretation of the meaning of multiple bands, however, is less straightforward, particularly if the protein is glycosylated (the bands can also be stained for... 

A clean-up process-scale RP-HPLC step has been introduced into production of human insulin prb. The C8 or C18 RP-HPLC column used displays an internal volume of 80 1 or more, and up to 1200 g of insulin may be loaded during a single purification run (Figure 11.4). Separation is achieved using an acidic (often acetic-acid-based) mobile phase (i.e. set at a pH value sufficiently below the insulin pi value of 5.3 in order to keep it fully in solution). The insulin is usually loaded in the water-rich acidic mobile phase, followed by gradient elution using acetonitrile (insulin typically elutes at 15-30 per cent acetonitrile). 

Quantitative studies comparing the relative abundances of proteins in different cellular states may be performed with MS. Methods such as 2D-GE have been utilized extensively with great success and differentially represent spots excised and then subjected to MS/MS for final identification of the differentially expressed proteins. 2D-GE requires approximately 50 pg of starting material and is limited by its bias toward high abundance proteins and propensity to detect proteins with extreme pi values. Furthermore, proteins at both extremes of molecular weight and those associated with membrane fractions are not well represented by 2D-GE.10... 

A pH gradient is produced by incorporating a mixture of Ampholines with appropriate p/ values in either a polyacrylamide slab or column. An acid is used at the anode and an alkali at the cathode, the pH of these being approximately the same as the pi values of the two extremes of the Ampholine range. Phosphoric acid and sodium hydroxide are suitable for wide range separations while various amino or carboxylic electrolytes are used for intermediate pH ranges. 

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