Manuel Sabés

Factors involved in the production of liposomes with a high-pressure homogenizer.

A variety of factors were evaluated in order to establish the characteristics of the liposomes obtained with a high-pressure homogenizer (Microfluidizer 110S). The experimental mean diameter of the liposomes and the width of their size distribution were correlated to surfaces calculated from the responses obtained by the combination of two groups of factors. First, the inlet pressure of the homogenizer, the times that the samples were processed (cycles) and the bulk ionic strength. Second, the phospholipid (P) and ethanol concentrations. The variation of the entrapped liposome volume upon the pressure and cycles was also studied. All the calculated surfaces are statistically significant and have a low S.E. of estimate. Mean liposome diameter decreases with increasing inlet pressure, number of cycles and ethanol concentration, and increases raising ionic strength. No P concentration effect was observed. The variation of the entrapped volume of liposomes upon the cycles and pressure has a similar behavior to the diameter. Within the studied variable range, microfluidization does not increase the P oxidation index, nor does it significantly alter the P concentration.

Fourth-derivative spectrophotometry of proteins

Fourth-derivative spectrophotometry offers several advantages over classical absorption or difference spectrophotometry in examining the characteristics of aromatic amino acids in proteins. The basic principles of the technique and its applications are outlined.

Induction of the blue form of bacteriohodopsin by low concentrations of sodium dodecyl sulfate

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (lambda max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.

Thr90 is a key residue of the bacteriorhodopsin proton pumping mechanism

Mutation of Thr90 to Ala has a profound effect on bacteriorhodopsin properties. T90A shows about 20% of the proton pumping efficiency of wild type, once reconstituted into liposomes. Mutation of Thr90 influences greatly the Schiff base/Asp85 environment, as demonstrated by altered λ max of 555 nm and pK a of Asp85 (about 1.3 pH units higher than wild type). Hydroxylamine accessibility is increased in both dark and light and differential scanning calorimetry and visible spectrophotometry show decreased thermal stability. These results suggest that Thr90 has an important structural role in both the unphotolysed bacteriorhodopsin and in the proton pumping mechanism.

Conformational Isomerizations of the Poly(dA-dT) and Poly(amino2dA-dT) Duplexes Involving the Unusual X-DNA Double Helix: A Fourth Derivative Spectrophotometric Study

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino2dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270-310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino2dA-dT), to n----pi electronic transitions is discussed.

DETECTION OF CHARGE-PERTURBATION OF TRYPTOPHAN RESIDUES BY FOURTH-DERIVATIVE SPECTROPHOTOMETRY

Abstract— Fourth‐derivative spectrophotometry has been applied to the analysis of the 1La O‐O band of the indole chromophore. Using several tryptophan models, it is shown that when this band is the most red‐shifted, it produces a minimum at about 300 nm in the fourth‐derivative spectrum. In proteins this red shift and the 300‐nm minimum results from a charge‐perturbation effect acting through space on the indole group of tryptophan residues. It is concluded that fourth‐derivative spectrophotometry can be used for monitoring the effect of charges on tryptophan residues

2-Hydroxy-5-nitrobenzyl bromide as a specific reagent for tryptophan residues in membrane proteins: bacteriorhodopsin as an example

The use of 2-hydroxy-5-nitrobenzyl bromide for the modification of tryptophan residues in integral membrane proteins is exemplified by its application to bacteriorhodopsin from Halobacterium halobium. Complete elimination of the unreacted reagent requires delipidation of the sample with detergents and posterior chromatography. This method also allows separation of the modified from the unmodified bacteriorhodopsin molecules. Modified molecules have lost the retinal, and are thus bleached, whereas the unmodified molecules appear to retain all the characteristics of solubilized native bacteriorhodopsin.