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The kinetics of the periodate oxidation of starch to dialdehyde starch have been studied thoroughly based on a reliable high-performance liquid chromatographic analysis. Investigation of the early stage of reaction has revealed that it proceeds initially following second-order kinetics. Later on, however, the resulting second-order model deviates from the experimental data. In order to get a model for the course of the total reaction, the kinetics have been approached from a different angle. Based on elementary kinetic principles, an appropriate model has been derived that can be used to simulate the oxidation process. An oxidation process performed semi-continuously has been modelled and compared with a batch process.

A method, using anion exchange chromatography and pulsed amperometric detection, is described for quantitative analysis of the oligosaccharides present in inulin. The analysis of a number averaged and molecular weight averaged degree of polymerisation and the dispersion of inulin and inulin fractions is given.

In this study several recently developed or modified tests for assessing biodegradability of water insoluble polymers in the aquatic environments are described and compared. By modifying the traditional single-phase closed bottle B.O.D. test, lower deviations and a higher rate of oxygen demand were obtained. Biological oxygen demands in the two-phase (semi-) closed bottle B.O.D. test correlated well with carbon conversion to inorganic gases under both, aerobic (modified Sturm test), and anaerobic circumstances (anaerobic digester test). The test methods presented in this report can be used to assess the extent and rate of biodegradation of a vast range of synthetic and natural insoluble polymers, varying from persistent (non-biodegradable) polyethylenes and slowly degradable modified starch materials, to fully biodegradable natural polymers of plant origin.

Effective procedures are reported for isolating and producing dimorphecolic acid (1) and its methyl ester (2) from Dimorphotheca pluvialis seed oil. The methods are suitable even for kilogram-scale production. The acid 1 was prepared by enzymatic hydrolysis of the oil and isolated by crystallization at low temperature. The ester (2) was obtained by methoxide-catalyzed transesterification of the glycerides followed by a countercurrent separation using four vessels and fourteen transfers with the two-phase system 90% aq. methanol-hexane. Methyl 9-oxo-10,12-E,E-octadecadienoate (3) (0.5 to 4%) was observed by GC and GC-MS and characterized by its reduction with sodium borohydride to racemic 2 and by its formation from 2 by Oppenauer oxidation. The complete assignment of the 1H and 13C-NMR signals of 1 and 3 has been carried out using 2D homonuclear and heteronuclear NMR experiments.

The effect of peptides on bilayer → non-bilayer phase transitions can be used as a tool to investigate the molecular aspects of peptide-lipid interactions. In this contribution the action on membranes of the peptide antibiotic gramicidin A and the bee venom component melittin are compared. Although the know structures and locations of these peptide upon membrane binding are very different, their actions on membranes show striking parallels. A general model is proposed that explains the seemingly complex peptide-lipid interactions by making use of simple concepts.

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.

A fast and direct method, based on infrared spectroscopy, for quantitative determination of starch short-range structure has been developed. The IR spectrum of starch is sensitive to changes in short-range structure in the C—C and C—O stretching region at 1300-800 cm−1. The IR absorbance band at 1047 cm−1 is sensitive to the amount of ordered or crystalline starch and the band at 1022 cm−1 is characteristic of amorphous starch. The ratios (R) of the heights of the bands at 1047 and 1022 cm−1, which expresses the amount of ordered starch to amorphous starch, and 1047 and 1035 cm−1, which is a measure of the amount of ordered starch, showed a linear regression with the amount of potato starch having B-type crystallinity as determined with wide-angle X-ray diffractometry. The IR spectrum and thus the short-range order is also sensitive to water content. In particular, the band at 994 cm−1, which is related to intramolecular hydrogen bonding of the hydroxyl group at C-6, is water sensitive. It is possible to quantify the IR data in terms of short-range order (SIR) over a range of 10–50% water. The method has been applied to quantify the changes in short-range structure during the melting of potato starch with 18 or 26% (w/w) water. The amount of short-range structure and the changes during melting in the (partially) destructured starch samples concur with differential scanning calorimetry and wide-angle X-ray diffractometry measurements.

Retrogradation kinetics for a potato starch-water system (10% w/w gel) was monitored by Fourier Transform Infrared spectroscopy and compared with waxy maize starch. The spectra showed the C-C and C-O stretching region (1300-800 cm−1) to be sensitive to the retrogradation process. A multi-stage process was observed during the retrogradation of potato starch and characterized as the formation of short- and long-range order. The first stage was characterized as the formation of helices and the fast formation of crystalline amylose regions. The second stage was described as the induction time for amylopectin helix aggregation. Stage three was described as the helix-helix aggregation and the crystallization of amylopectin. The overall-first order calculated rate constant of potato starch was (9.6±1.4) 10− 3h−1. The calculated rate constant were in agreement with the known difference in retrogradation kinetics of waxy maize and potato starch. The effects were explained by the differences in retrogradation rate of amylopectin and amylose. Potato starch consists of amylose as well as amylopectin. Whereas amylose crystallization occurs within a few hours, amylopectin crystallization is slow and takes a few weeks.

The influence of glycerol on the retrogradation kinetics for waxy maize starch-water systems was monitored by Fourier transform infra-red spectroscopy. The spectra showed the C---C and C---O stretching region (1300-800 cm−1) to be sensitive to the retrogradation process. A multistage kinetic process in terms of structural changes on a molecular level was observed during the retrogradation of waxy maize in a 10% (w/w) gel. For the first two stages (formation of helices and induction time for helix aggregation) no significant kinetic differences were observed in the gels with various glycerol contents. Stage three is described as the primary aggregation and crystallization step. In the final stage, syneresis of water occurs. The calculated rate constants clearly show decreasing retrogradation of waxy maize starch with increasing glycerol content. The effects are explained in terms of starch-glycerol interactions and stabilization of water resulting in a decreased mobility of the starch chains.

The 1 H chemical shifts of sucrose, 1-kestose and nystose and the 13 C signals of nystose have been completely assigned by using 1D- and 2D-NMR techniques. Accurate values of 3 J H,H coupling constants were obtained for these molecules.

We studied the differential effect of tryptophan-N-formylated gramicidin on uninfected and Plasmodium falciparum-infected erythrocytes. Trp-N-formylated gramicidin induces a much faster leakage of K+ from infected cells than from uninfected cell whereas, and at an even lower concentration, gramicidin A' causes a rapid K+ leakage from both uninfected and infected cells. We also studied the effect of Trp-N-formylated gramicidin and gramicidin A' incorporated in liposomes on the growth of Plasmodium falciparum in an in vitro culture. Incorporation of Trp-N-formylated gramicidin in the membranes of so-called 'stealth' vesicles strongly decreases the concentration needed to induce 50% inhibition of parasite growth. Moreover, no decrease in the K+ content of uninfected cells was observed when cells were exposed to liposome-incorporated Trp-N-formylated gramicidin at a concentration which causes full inhibition of parasite growth. These observations strongly suggest that Trp-N-formylated gramicidin incorporated in 'stealth' vesicles ends up specifically in the infected cell, thereby inhibiting the growth of the growth of the malaria parasite.

Lipids in isolated form show polymorphism. Depending on the type of lipid and experimental conditions, different macromolecular aggregate structures are formed. for membrane lipids hydrated in excess water, under physiological conditions of ionic strength, pH and temperature, three major liquid-crystalline phases are observed. These are: the lamellar phase, the inverted (type II) hexagonal phase (Fig. 1), and the normal (type I) micellar phase. The shape-structure concept of lipid polymorphism as originally proposed by Israelachvili et al. (1976) and adapted by Cullis and De Kruijff (1979) explains remarkably well most of the current data on lipid polymorphism (Fig. 2). If the surface area in the head group region matches that of the hydrocarbon region (such as in phosphatidylcholines) the molecule has an overall cylindrical shape and prefers to organize in extended bilayers. When the head group area exceeds the hydrocarbon area, as for instance in case of detergent-like lipids such as lysophosphatidylcholines, the molecule on average is cone shaped and prefers an organization with a convex (type I) surface curvature such as found in micelles or in the related normal (type I) hexagonal phase occurring for these lipids at low water content. Phosphatidylethanolamines are key representatives of the opposite case of an excess hydrocarbon area. This results in the concave (type II) surface curvature as found in the HII phase. Head group hydration and inter- and intramolecular interactions are important determinants for the shape of the lipid molecules in the shape-structure concept. The strictiy regulated (Goldfine et al., 1987; Wieslander et al., 1981) presence of large amounts of type II lipids (lipids which in hydration prefer to organize in type II structures) and their abundance in virtually all biomembranes suggests important structural and functional roles for these types of lipids.

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1:10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N- succinylgramicidin and O- succinylgramicidin . The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.

Incorporation of the channel-forming antibiotic gramicidin into the membrane of human erythrocytes highly (up to 30-fold) enhances rates of reorientation (flip) of lysophosphatidylcholine and palmitoylcarnitine to the inner membrane layer after their primary incorporation into the outer layer. Despite the high increase of flip rates by gramicidin, the asymmetric orientation of the inner membrane layer phospholipids phosphatidylethanolamine and phosphatidylserine is stable as demonstrated by the lack of accessibility of these lipids toward cleavage by exogenous phospholipase A2. On the other hand, gramicidin enhances the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2, which indicates changes in the packing of phosphatidylcholine following gramicidin binding. The increase of flip becomes detectable when about 10(5) copies of gramicidin per cell have been bound (gramicidin to membrane phospholipid ratio of 1:2000). This is a 1000-fold higher concentration than that required for an increase of K+ permeability mediated by the gramicidin channel. Acceleration of flip is thus not simply correlated with channel formation. The enhancement of flip is markedly dependent on structural details of gramicidin. Formylation of its four tryptophan residues abolishes the effect. Even at high concentrations of formylated gramicidin at which the extents of binding of native and of formylated gramicidin to the membrane are comparable, no flip acceleration is produced. Enhancement of flip by gramicidin occurs after a temperature-dependent lag phase. At 37 degrees C, flip rates begin to increase within a few minutes and at 25 degrees C, only after 3 h. This lag phase is most likely not due to limitations by the rate of binding of gramicidin to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of cholesterol incorporation on gramicidin-induced hexagonal H II phase formation in different phosphatidylcholine model systems was investigated by 31 P- and 2 H-NMR, small-angle X-ray diffraction and differential scanning calorimetry. In liquid-crystalline distearoylphosphatidylcholine systems cholesterol inhibits gramicidin-induced H II phase formation. In dioleoylphosphatidylcholine the opposite effect is observed. Cholesterol appears to preferentially interact with gramicidin under liquid-crystalline conditions in both systems. Two phenomena that had been reported for gramicidin-treated erythrocyte membranes and derived liposomes (Tournois, H., Leunissen-Bijvelt, J., Haest, C.W.M., De Gier, J. and De Kruijff, B. (1987) Biochemistry, 26, 6613–6621) could also be observed in more simple dioleoylphosphatidylcholine-gramicidin-cholesterol systems. These are (i) an increase in tube diameter in the gramicidin-induced H II phase with increasing temperature, which is ascribed to the presence of cholesterol in this phase, and (ii) the loss of the hexagonal H II phase related 31 P-NMR line shape at lower temperatures despite the presence of this phase as demonstrated with X-ray diffraction. This latter phenomenon appears to be due to restrictions in the rate of lateral diffusion of the phospholipids around the H II tubes due to the presence of gramicidin.

It is shown by 31P-NMR and small angle X-ray scattering that induction of an hexagonal HII phase in dioleoylphosphatidylcholine model membranes by external addition of gramicidin A' depends on the solvent which is used to solubilize the peptide. Addition of gramicidin from dimethylsulfoxide or trifluoroethanol solution leads to HII phase formation whereas addition of the peptide from ethanol does not. This solvent dependence is shown by circular dichroism to be correlated with the peptide conformation. The channel conformation appears to be responsible for HII phase formation by gramicidin.

Gramicidin is a potent modulator of long-range membrane lipid organization and therefore a useful model to understand aspects of lipid polymorphism in relation to the structure and function of biological membranes. In this article we will review the peptide’s lipid structure-modulating activity in relation to recent studies on the lipid flip-flop enhancing ability of gramicidin in erythrocyte membranes. Special emphasis will be placed on the essential role of the conformation of gramicidin and its aggregational behavior in these events. The relationships between the other known properties of gramicidin, e.g., channel formation and involvement of gene regulation, are indicated. It is suggested that the unique concentration of the four tryptophans towards the C-terminus is the structural link between these different functional properties of the peptide.

Gramicidin films at the air/water interface are shown to exhibit a phase transition at 225 A2/molecule which might be caused by either cluster formation, reorientation of molecules, conformational changes or multilayer formation. It is further shown that coupling of a charged group on either NH2- or COOH-terminus or elongation of the peptide by two amino acids, only slightly affects the surface area characteristics whereas modification of the tryptophans or even replacement of a single tryptophan by phenylalanine leads to drastic alterations in the surface-area characteristics and a (partial) loss of the phase transition demonstrating that the tryptophans play an important role in the interfacial behavior of gramicidin. The lack of a solvent history effect on the interfacial behavior indicates a rapid conformational interconversion of the peptide at the air/water interface. Gramicidin in mixtures with dioleoylphosphatidylcholine and lysopalmitoylphosphatidylcholine shows a condensing effect whereas gramicidin shows ideal mixing with dioleoylphosphatidylethanolamine. The condensing effect most likely is related to the aggregational state of the peptides which is different in phosphatidylcholines and phosphatidylethanolamines.

Using 31P nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and freeze-fracture electron microscopic (FFEM) techniques, it is shown that gramicidin induces a hexagonal HII phase not only in liposomes prepared from total lipids extracted from human erythrocytes but also in isolated human erythrocyte membranes (white ghosts). A 37 degrees C, HII phase formation is detected at a gramicidin to phospholipid molar ratio exceeding 1:80. At a molar ratio of 1:5, about 30% of the phospholipid is organized in the HII phase. The gramicidin-induced HII phase exhibits a very small 31P chemical shift anisotropy [(CSA) approximately 10 +/- 1 ppm], indicating decreased head-group order, and it displays a temperature-dependent increase in tube diameter from 60.2 A at 4 degrees C to 64.2 A at 37 degrees C in ghosts and from 62.8 to 69.4 A at 37 degrees C in total lipid extracts, both in the presence of 1 mol of gramicidin/10 mol of phospholipid. This anomalous temperature-dependent behavior is probably due to the presence of cholesterol. 31P NMR data indicate that the HII phase formation by gramicidin is temperature dependent and show the gradual disappearance of the HII phase at low temperatures (less than 20 degrees C), resulting in a bilayer type of 31P NMR line shape at 4 degrees C, whereas SAXS and FFEM data suggest equal amounts of HII phases at all temperatures. This apparent discrepancy is probably the result of a decrease in the rate of lateral diffusion of the membrane phospholipids which leads to incomplete averaging of the 31P CSA in the HII phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal HII phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (beta 6.3) conformation of the peptide is essential for all three membrane perturbing effects.