John C. Allen

The use of zwitterionic surfactants in the agarose chromatography of biological membranes

The use of surfactants in the separation and analysis of the components of biological membranes is widespread [ 11. Several groups have investigated the agarose chromatography of surfactant-treated membrane material, using an eluant which itself contains the surfactant; such studies have been made on the plasma membrane of rat liver cells [2], human erythrocyte membranes [3-71, nuclear membranes [8], and the milk fat-globtl.le membrane [9,10]. The chemical classes of surfactant used in such work have been limited: sodium dodecyl sulphate [6,10], sodium deoxycholate [3], Triton X-100 (p-isooctylphenoxypolyethoxyethanol) [4,5,7,8] or a mixture of these [2,9] being most commonty employed. In the course of an investigation of the milk fatglobule membrane, a study was made of the effects on the membrane of a number of unusual surfactants; these included the zwitte.ionic alkylbetaine, Empigen BB*:

The reactivities of furocoumarin excited states with DNA in solution. A laser flash photolysis and fluorescence study.

The effect of DNA on both the fluorescence emission spectra and yields and lifetimes of the triplet stae of psoralen and 8-methoxypsoralen in aqueous solution has been determined. The changes in the fluorescence spectra are similar in nature for both of these furocoumarins and are attributed to binding of the drug to DNA. The yield of the 8-methoxypsoralen triplet state when bound to DNA was found to be similar, if not identical, to that measured in the absence of DNA. This contrasts sharply with data obtained for psoralen from which it is concluded that either the yield of bound psoralen triplet states is very low, if not zero, or that the lifetime of such species is less than 50 ns. The relevance of this data to the molecular basis of skin photosensitisation by furocoumarins is discussed.

An evaluation of the oral administration of commercial and fractionated heparin samples in rats

The oral absorption of the anticoagulant, heparin, has been studied in rats Heparin fractions from various commercial sources and with differences in molecular weight, anticoagulant activity and the type of counterions present have been tested. The results show that none of the heparin samples were absorbed into the blood system under the experimental conditions employed. Further work by administering heparins in non-toxic acids and complexed to spermine and lysine did not produce any absorbability of the drug. The experiments also revealed inherent changes in clotting times of rat plasma related to the animals age between 8 and 16 weeks. For the activated partial thromboplastin time assay, clotting time (s) = 2.105 × age (weeks) + 7.18(P < 0.001) and for the thrombin- fibrinogen time assay, clotting time (s) = 0.483 × age (weeks) + 8.67 (P < 0.01).

The influence of micelle formation on lipoxygenase kinetics

The effects of a series of n-alcohols and n-carboxylic acids on lipoxygenase activity was studied. It was shown that to a large extent the effects of these compounds could be ascribed to physiochemical interaction with the substrate solution rather than a direct action on the enzyme itself. The effect of better substrate analogues such as stearate and oleate could also be ascribed to this effect. A type-2 lipoxygenase was found to have a very unusual velocity-substrate relationship which could be normalized by addition of calcium chloride in amounts stoichiometric with the substrate. An excess of calcium inhibited the enzyme. By comparison of results with linoleoyl sulphate/linoleoyl alcohol mixed micelles, an explanation for this unusual velocity-substrate activity is presented.

An investigation of calcium ion binding to heparins

Using the line charge model of Manning and, where appropriate, a form of the empirical corrections to his limiting laws proposed by Wells, we have investigated the ion condensation phenomenon for four pure calcium heparin preparations and similar Ca-heparin/CaCl2 mixtures in aqueous solution. This has been expressed in terms of the single ion activity coefficient of the total calcium counter ion present. The results found for the single counter ion activity coefficient of pure Ca-heparin agree moderately well with the limiting law of Manning for a pure divalent counter ion polyanion salt in dilute solution. The results for Ca-heparin/CaCl2 mixtures when corrected for small ion-small ion interactions give a surprisingly good fit to the theoretical counter ion activity coefficient for a divalent counter ion/divalent cation simple electrolyte system, for values of X (X = the polyanion/co-ion ratio) up to 4, with slight deviations for values of X > 4. In the mixtures with the largest excess of polyelectrolyte, the single ion activity coefficients of the Ca2+ ion are in excess of the value predicted by Mannings theory. Ion exchange of sodium heparins to give the Ca-heparin samples used in the work did not appear to cause regular changes in the anticoagulant activity.

The oxidation of lipids by components of bovine milk-fat globule membrane

Bovine milk-fat globule membrane was solubilized with a zwitterionic surfactant and subjected to chromatography on agarose, with the surfactant in the eluant. Fractions were tested for their effects on the oxidation of buffered linoleate. The maximum oxidative capability was greatly enhanced by the addition of Cu, and became associated with the phospholipids. Further chromatography of the retarded protein peak from agarose on Sephadex G-200, again in the presence of surfactant, gave 2 protein peaks. Oxidative effectiveness resided almost entirely in the first peak, which was devoid of phospholipid, but high in xanthine oxidase activity. This fraction was subjected to isoelectric focusing, and the xanthine oxidase from this was highly pro-oxidative. Furthermore, its oxidative capability was almost doubled on heat treatment.

Some physicochemical and biological characteristics of heparin fractions prepared by gel chromatography

Abstract Heparins of defined molecular weights fractionated on gel chromatography in the presence of salt, have been assayed for their anticoagulant (B.P.) and anti-Xa activities, protamine neutralization and equivalent weight values as well as their abilities to release lipoprotein lipase. The anticoagulant activity and lipoprotein lipase releasing ability appears to decrease with molecular weight. In contrast the anti-Xa activity of the low molecular weight fraction showed an increase. No discernable trend was noted for equivalent weight and protamine neutralization values of the fractions.