Reverse micelles in protein separation: the use of silica for the back-transfer process

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative "back extraction" procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (alpha-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a alpha-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. (c) 1993 John Wiley & Sons, Inc.     

The consequences of demographic reduction and genetic depletion in the endangered Florida panther

The Florida panther has recently suffered severe range and demographic contraction, leaving a remarkably low level of genetic diversity. This exerts a severe fitness cost, manifested by spermatozoal defects, cryptorchidism, cardiac abnormalities and infectious diseases that threaten the survival of the subspecies.     

Alteration by dinitrocresol of pathways for glucose oxidation in eggs of arbacia punctulata

1. The C¹⁴O₂ production by Arbacia eggs and embryos from glucose-1-C¹⁴, glucose-2-C¹⁴, and glucose-6-C¹⁴ has been measured without and with dinitrocresol in the incubation medium. In the absence of the dinitrocresol, the C¹⁴O₂ production from glucose-1-C¹⁴ is more rapid than from glucose-2-C¹⁴ and much more rapid than from glucose-6-C¹⁴; this, together with previous findings, indicates that glucose is utilized in Arbacia eggs predominantly via the TPN shunt rather than via the aldolase step of the glycolytic pathway. In the presence of the dinitrocresol, C¹⁴O₂ from glucose-6-C¹⁴ approaches that from glucose-1-C¹⁴, indicating that, in the presence of this reagent, glucose utilization is diverted from the shunt to the glycolytic pathway. 2. Incorporation of C¹⁴ from glucose labelled in the 1-, 2-, or 6- positions into other metabolic products of the eggs and embryos is also inhibited by dinitrocresol, particularly incorporation into the acid-insoluble fraction containing nucleoproteins.     

The effect of changes of environment on the electrical and ionic pattern of muscle

The resting and action potentials of sartorius muscles of the toad, Bufo marinus, have been measured under varying conditions of external environment. At the same time, analyses for Na(+) and K(+) content were carried out. There was a slight elevation of 2 mv. when the measurements were made in phosphate-Ringer instead of in bicarbonate-Ringer. The R.P. was independent of the hydrogen ion concentration between pH 6.5 and 8.5, although at these pH's there was marked alteration in the level of Na(+) and K(+) in the muscle. Alteration of the external K(+) level between 0 and 50 m.eq./liter has little influence on the internal K(+) concentration. When the log of the external K(+) concentration is plotted against the R.P. there is not a linear relationship until the external K(+) is raised above 12 m.eq./liter, at which point the cell is unexcitable. Above this value a straight line with a slope of 58 mv. per ten-fold change in concentration is obtained, but the absolute values at any point are about 35 per cent higher than those which would be given by the Nernst equation. Alteration of the external Na(+) level within a range of 45 to 650 m.eq./liter resulted in marked changes in the internal Na(+) content, without, however, having any effect on the ratio Na(+) (out)/Na(+) (in). This ratio has remained at about 3 in spite of marked fluctuations in the absolute value of the internal and external Na(+) levels. When the Na(+) level is lowered there is a decrease in the height of the action potential although there is no alteration in the ratio Na(+) (out)/Na(+) (in). As the Na(+) level is raised the height of the action potential is not affected even in the presence of a fivefold increase in Na(+) in the Ringer. The results do not support the conclusion that the bioelectric potentials can be calculated from the ionic ratios by means of simple physical chemical hypotheses such as the Nernst or Goldman equations. The maintenance of the normal K(+) content of the cell cannot be accounted for by a Donnan mechanism. No definite evidence has been produced to explain the mechanism of a Na(+) "pump." In other words, the concept of a Na(+) pump requires that there shall be a physico- or organochemical mechanism which will distinguish between Na(+) and K(+) (or other) ions. There is evidence that Na(+) can be extruded against a concentration gradient. On the other hand the cell is able to maintain a constant ratio of external to internal Na(+) even when the cell has been severely damaged by very high external Na(+) levels.