Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.     

pK determinations via pH-mobility curves obtained by isoelectric focusing-electrophoresis: Theory and experimental verification

Basic equations have been derived linking the electrophoretic migration in a stationary pH gradient of simple, singly charged cations or anions and of mono- mono- valent ampholytes with the pKs of their ionizable groups. In the case of diprotic ampholytes, an equation and a curve are described calculating a correction factor to be applied to the mobility measurements, accounting for the influence of the opposite charge species on the mobility curve of the ion being measured. This correction factor is a function of ΔpK and increases exponentially with decreasing values of ΔpK. These theoretical considerations have been experimentally verified by running pH-mobility curves of colored compounds, such as methyl red, neutral red and dexorubicin. The pKs thus measured were in excellent agreement with the pKs obtained independently by spectrophotometric titrations.     

Transient state isoelectric focusing. Digital measurement of zone position, zone area, segmental pH gradient, and isoelectric point as a function of time

Transient state isoelectric focusing (TRANSIF) is a kinetic method which offers quantitative information about relevant parameters pertaining both to methodological aspects and to the physical characterization of amphoteric molecules. TRANSIF data are obtained with an improved scanning isoelectric focusing assembly coupled to an on-line digital data acquisition and processing system which is used to continuously record changes in peak position ($\text{x}$), peak area, segmental pH gradient ($\text{Δ(pH)}\text{Δx}$), and isoelectric point (pI) of proteins during focusing. This improved apparatus has made it feasible to follow the temporal stability of an isoelectric focusing system in a quantitative fashion.     

Electrophoresis, crossed immunoelectrophoresis, and isoelectric focusing in agarose gels with reduced electroendosmotic flow

An ion exchange method using QAE-Sephadex for preparation of agarose with a low electroendosmotic flow and reduced adsorption properties is described. The successful use of such agarose for the separation of highly cationic proteins is illustrated.A method for isoelectric focusing of proteins in gels made from a mixture of purified agarose and a water-soluble non-cross-linked acrylamide polymer is described. This method can be combined with immunochemical identification by electrophoresis of the separated components into antibody-containing agarose gels, also containing such a polymer of acrylamide.     

Characteristics of the isoelectric focusing procedure: Importance of column size,pH, and protein-protein interactions

Isoelectric focusing (IEF) on 110- and 440-ml columns can result in the loss of enzyme activity. Such losses can be reduced or eliminated by focusing on 20-ml columns. Artifacts which arise during the IEF procedure may result from protein-protein interaction or from the interaction of pH with other sources of artifacts. The reasons for greater loss of activity on large columns and the mechanism of artifacts due to pH and protein-protein interactions are discussed.     

Amidosulfobetaines, a family of detergents with improved solubilization properties: application for isoelectric focusing under denaturing conditions

The synthesis of a family of new detergents and their use for isoelectric focusing are described. These detergents differ from the conventional sulfobetaines by the presence of an amido group bridging the hydrophobic linear tail and the polar head, which increases their water solubility and urea tolerance considerably. Four different linear alkyl hydrophobic tails were tested, together with three different polar heads. The solubilization of red blood cell ghosts by the 12 resulting detergent performances was evaluated both in solution and in isoelectric focusing runs. Six chemicals gave performances equal or better than those of conventional detergents and could replace them for general use in focusing experiments. In addition, the synthesis of a whole range of compounds allowed valuable insights in the empirical understanding of protein-detergent interactions, which are extensively discussed.     

Transient state isoelectric focusing: effects of zone load and carrier ampholyte concentration on the kinetics of defocusing and refocusing in sucrose density gradients

The kinetics of focusing, defocusing, and refocusing of l-histidyl-l-tyrosine in a sucrose density gradient have been studied utilizing a special apparatus for repetitive scanning of the isoelectric focusing column, employing uv absorption optics and a digital data acquisition system. Starting from a uniform or triangular concentration profile, the band is “focused” and a nearly linear pH gradient is formed during initial focusing. The electrical field is then abolished and free diffusion occurs. The electrical field is then reapplied (“refocusing”) and the band is allowed to sharpen, presumably reapproaching a steady state. The band width was measured quantitatively as the second moment about the mean (square of the standard deviation, σ²). In theory, measurements of σ² versus time permit the estimation of the apparent diffusion coefficient (D) and the isoelectric focusing parameter (pE). If the electrical field strength E and the pH gradient, $\text{d(}\text{pH}\text{)}\text{dx}$, were also measured, then one could calculate the slope of the pH mobility curve of the protein $\text{dM}\text{d(}\text{pH}\text{)}$ evaluated at the isoelectric point. D can be measured during the defocusing stage, and $\text{pE,}\text{D}\text{pE}\text{,}\text{or}\text{D}$ can be measured during focusing or refocusing. Several limitations and difficulties in the verification of this theory have been encountered: First, the apparent diffusion coefficient depends on zone load in approximately a linear fashion. Accordingly, it is necessary to measure D at several zone loads, and then extrapolate to zero load by linear regression techniques. Second, the ampholyte concentration has a marked effect on both D and pE. Here we have no a priori reason to extrapolate to zero ampholyte concentration. Also, at present we have no satisfactory method for measurement of E. These preliminary studies should be helpful in indicating further directions for experimental refinement and for generalization of theory.     

Chemotaxonomy of Hypericum genus from Portugal: Geographical distribution and essential oils composition of Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum

The geographical distribution and analysis of the essential oils of species from three sections of Hypericum L. (Guttiferae/Clusiaceae/Hypericaceae) from Portugal are presented. Hypericum perfoliatum (section Drosocarpium) grows wild in the centre and south of Portugal; Hypericum humifusum and Hypericum linarifolium are both from section Oligostema, the former occurring throughout the country, while the second is distributed mainly in the north and centre; Hypericum pulchrum (section Taeniocarpium) is confined to the littoral north of Portugal. The essential oils were obtained by distillation–extraction, hydrodistillation and distillation in a modified Marcusson apparatus from the dried aerial parts of the different populations and were analysed by GC and GC–MS. Monoterpene hydrocarbons constituted the main fraction in all oils (43–69%, 53–85%, 28–45% and 48–65% for H. perfoliatum, H. humifusum, H. linarifolium and H. pulchrum, respectively). Sesquiterpene hydrocarbons (2–13%, 6–18%, 21–27% and 16–18%, respectively) and a third fraction of non-terpenic compounds (20–29%, 3–16%, 2–14% and 5–11%, respectively) from the four species attained relatively high amounts in all oils. Within each species, no major differences were detected in the essential oil composition, despite the fact that different locations, phenological phases and extraction methodologies were used. Notwithstanding the dominance of α-pinene in all four species' oils, cluster and principal components analysis on the identified components showed that the range of α-pinene, β-pinene and n-nonane supported a separation of the four species. The essential oil composition of the four species showed some qualitative resemblances, which correlate well with the taxonomical classification based on morphological characters.     

Determination of isoelectric points in thin-layer isoelectric focusing: The importance of attaining the steady state and the role of CO2 interference

Isoelectric points differing by 1 to 2 pH units are measured for horseradish peroxidase and lactoperoxidase depending upon the technique of isoelectric focusing, namely, the density gradient technique or systems stabilized by either granulated (Sephadex, Bio-Gel) or compact polyacrylamide gels. Conditions standardized for the determination of pI values of selected pH marker proteins proved inadequate for the predominant isoenzyme of horseradish peroxidase which requires an excessively long focusing time to attain the steady state. Carbon dioxide interferes with the determination of pI values >8.2 to 8.3. Thin-layer isoelectric focusing in a CO₂-free atmosphere followed by pH measurements also in a CO₂-free atmosphere, yields for alkaline marker proteins and the predominant peroxidase isoenzyme, pI values in excellent agreement with these found by the density gradient technique. The isoionic point of the predominant peroxidase isoenzyme determined by ion exchange desalting is identical with the isoelectric point found by density gradient and thin-layer isoelectric focusing in a CO₂-free atmosphere.     

Peptide mapping of bovine and chicken cytochrome c by capillary isoelectric focusing with universal concentration gradient imaging

Capillary isoelectric focusing with universal concentration gradient imaging detection was used to separate and detect tryptic peptides from bovine and chicken cytochrome c. For a desalted sample of peptide angiotensin 2, the isoelectric point (pI) measured by the instrument agreed well with the pI calculated from amino acid pK values. For the cytochrome digests, correlations between measured and calculated pI values were imprecise because peak positions shifted slightly from test to test. This problem is thought to be caused by the inefficient desalting process used on the samples, leaving salt residues which caused distortion in the pH gradient during the focusing process. However, this system differentiated between the two cytochrome c's. The concentration gradient imaging detected peptides which contain no tyrosine and no tryptophan amino acids, which a UV absorption detector operating at 280 nm could not. The separation and detection steps took only 5–7 min because no mobilization was necessary after the focusing process.     

Interaction strength between a predator and dangerous prey: Sinistrofulgur predation on Mercenaria

The lack of direct empirical evidence of predator evolution in response to prey adaptation is a fundamental weakness of the arms race analogy of predator-prey coevolution. I examined the interaction between the predatory busyconine whelk Sinistrofulgur sinistrum and its bivalve prey Mercenaria mercenaria to evaluate whether reciprocal adaptation was likely in this predator-prey system. Thick-lipped whelks use their shell lip to chip open the shell of their prey, often resulting in breakage to their own shell. Thus, hard-shelled prey, such as Mercenaria, may be considered dangerous because they are able to inflict damage to the predator as a consequence of the interaction. The strength of interaction between whelks and their bivalve prey was viewed by regressing predator performance (the incidence of shell breakage in encounters with prey) on prey phenotype (a function of size). Interaction with Mercenaria of varying sizes has strong and predictable consequences (r²=0.946; p=0.028) for Sinistrofulgur. Predators that select large, thick bivalve prey increase the likelihood that their shell lip will be broken in the process of attempting to open their prey. Ecological consequences of feeding-induced breakage may include reduced growth rate, reproductive success, and survivorship. These results suggest that natural selection should favor predator phenotypes that reduce feeding-induced breakage when interactions with damage-inducing prey occur.     

Deficient fumarase activity in an infant with fumaricacidemia and its distribution between the different forms of the enzyme seen on isoelectric focusing.

A male infant, whose parents were first cousins, presented at 6 mo of age with hypotonia, microcephaly, and delayed development. He was found to have large amounts of fumaric and succinic acids present in the urine. In lysed cultured skin-fibroblast preparations, the activity of fumarase was found to be 22.7% of that in controls. Cell fractionation by homogenization and by digitonin treatment indicated that the residual activity in the cells of the patient was primarily located in the mitochondrial fraction rather than in the cytosolic fraction. Isoelectric focusing of fibroblast extracts showed that six bands of fumarase activity were discernible in control cell lines, two of them cytosolic with pI's of 5.53 and 5.60 and four of them mitochondrial with a pI of 5.65-6.8. In contrast, isoelectric focusing of fibroblast extracts from the fumarase-deficient patient showed only a single band of activity with a pI corresponding to the mitochondrial type seen in the controls. Immunoprecipitation of proteins with rabbit antifumarase antibody in (35S)-methionine-labeled fibroblasts indicated that a protein of correct size (Mr = 44,000 daltons) corresponding to fumarase was synthesized in similar amounts in both the patients and controls. It is proposed that in the patient's cells a single active species of fumarase that is mitochondrial in location is synthesized. Since it is known that mitochondrial and cytosolic fumarases are encoded by the same gene but differ slightly in amino acid sequence, it is possible that a point mutation might explain these findings.     

Physicochemical methods for predicting the biological potency of recombinant follicle stimulating hormone: an international collaborative study of isoelectric focusing and capillary zone electrophoresis.

Two methods for predicting the specific in vivo bioactivity of recombinant follicle stimulating hormone (FSH), based on quantitative measures of isoform distribution by isoelectric focusing (IEF)(1) and by capillary zone electrophoresis (CZE)(2) respectively, have been subjected to an international collaborative study by six laboratories from six countries. Both methods were used to estimate the predicted bioactivities of four preparations of follitropin beta, coded FSH A-D, differing widely in their isoform compositions and specific bioactivities. The mean predicted estimate of potency by IEF and CZE for each FSH preparation by each laboratory was within 80-125% of its potency estimated by bioassay, except for the mean estimates by CZE of that for FSH A by one laboratory and of that of FSH D by another. Each of the six laboratories using the IEF method, and each of the five laboratories using the CZE method were able to rank these FSHs according to their bioactivities, namely FSH B>FSH C>FSH A>FSH D. All laboratories were able to use both IEF and CZE to discriminate between FSH A and C, with bioactivities within 76-132% of one another. Four of six laboratories were able to use IEF, and two of five laboratories were able to use CZE, to discriminate between FSH B and C, with bioactivities within 89-112% of one another. This suggests that the accuracy and precision of both these methods should be sufficient to discriminate between FSHs which would meet or fail European Pharmacopoeia requirements for this type of hormone, since these stipulate that estimates of potency should fall between 80-125% of its stated potency. Using in most cases duplicate estimates in two independent assays, and excluding Laboratory 4, the pooled intra-laboratory geometric coefficient of variation (GCV) was about 4% for both IEF and CZE, and the inter-laboratory GCV was about 7% for IEF and about 10% for CZE. The use of one FSH preparation as a standard, with its specific activity as an assigned value, reduced the inter-laboratory variability of estimates for the remaining FSHs by both methods. This increased the accuracy of the predicted estimates of bioactivity for these remaining FSHs in terms of their approximation to the values for their bioactivities estimated by bioassay. These data therefore suggest that both these methods, and particularly IEF, are sufficiently accurate, precise and robust to be used for predicting the bioactivity of batches of follitropin beta, and especially if used with a standard preparation.     

Some effects of time,temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae

Dalgliesh R. J. and Stewart N. P. 1982. Some effects of time, temperature and feeding on infection rates with Babesia bovis and Babesia bigemina in Boophilus microplus larvae. International Journal for Parasitology12: 323–326. Percentages of larval ticks in which Babesia bovis and B. bigemina parasites could be detected (infection rates) were determined after the larvae had been exposed to temperatures between 9°C and 27°C for periods of 1–35 days and then either fed on calves or heated at 37°C to stimulate babesial development. Infection rates with both species increased during 2–4 weeks after the larvae hatched, regardless of the temperature of exposure. Infection rates with B. bovis were higher after exposure of larvae to 14°C than to 27°C. This effect was less pronounced with B. bigemina. Infection rates were higher in fed larvae than in unfed, ‘heat stimulated’ larvae. The findings indicate that infected larval ticks become more efficient vectors of Babesia during the first 2–4 weeks after hatching and that repeated sampling of a tick population is necessary to determine valid infection rates.     

A highly crosslinked, transparent polyacrylamide gel with improved mechanical stability for use in isoelectric focusing and isotachophoresis.

A safe and convenient means of monitoring very low rates of flow of sterile microbiological nutrient medium is described.     

Cochocelis distribution on the firth of clyde: estimates of the lower limits of the photic zone

A series of transects in the Firth of Clyde have shown that filaments of Conchocelis in shell substrata are always the deepest growing red algae. The abundance of calcareous material on the sea bed and its invariable association with the perennial Conchocellis filaments offers a reliable means of estimating photic limits. Some interactions of grazing molluscs with the shell-boring organism have been observed. Some of the pitfalls to be encountered in field determinationsof Conchocelis and its consequent use as an indicator organism are described.     

First records of flower biology and pollination in African Annonaceae: Isolona, Piptostigma, Uvariodendron, Monodora and Uvariopsis

Seven species from five genera of Annonaceae were studied with regard to their flower biology and pollination in the Southwest Province of Cameroon, West Africa. They have protogynous hermaphroditic flowers, with exception of Uvariopsis species, which are monoecious. Fused petals of Isolona campanulata remain apically spreading and open during anthesis but form a deep basal urceolate tube around the reproductive organs. At anthesis the yellow pendent flowers emit a fruit-like scent and attracted small beetles, the likely pollinators. Piptostigma sp. flowers also emit a fruit-like scent but provide a closed pollination chamber formed by the three inner petals. Small staphylinid beetles attracted during the female stage of anthesis are released from the flowers at the end of the male stage 2-3 days later. Both species have diurnal anthesis, attracting and releasing the flower visitors during daytime. In contrast, Uvariodendron connivens and U. calophyllum have nocturnal anthesis with floral thermogenesis, produce spicy, aromatic and fruity scents and attract large Scarabaeidae beetles, the pollinators, along with many curculionid beetles, which were principally predators of the thick petals. The very large flowers of Monodora tenuifolia have yellowish petals which are spotted with dark red markings. Together with the sweetish, slightly disagreeable scent the flowers attract flies, principally dung flies. The two investigated Uvariopsis species are monoecious with pistillate and staminate flowers being functional at the same time. The violet red flowers of U. bakeriana visually seem to mimic the fruiting body of certain stinkhorn fungi (Phallaceae) although without producing their strong unpleasant carcass stench. Flower-visiting dung flies were rare. Conversely, U. congolana has a strong fungus-like scent, its flowers are presented at litter height and dung flies living in the litter were the flower visitors, albeit sporadic. The 4-5 days lasting anthesis of both Uvariopsis species appears to be an evolutionary consequence of their diffuse pollinator spectra. The studied African Annonaceae therefore have either cantharophilous or myiophilous/sapromyiophilous flowers with, in part, respectively, remarkably long anthesis, thermogenesis, and widely open, large flowers - all attributes unknown or rare in the hitherto better studied Neotropical Annonaceae.