Further results on lipase-colipase interactions studied by affinity chromatography

Affinity chromatography of lipase on a colipase-coupled gel was studied in the present paper. The elution volume of the associable lipase increased when the loaded amount decreased. A KD value of 1.9 X 10(-6) M at pH 6.2 was thus deduced. A minimum value of 1.5 X 10(-6) M was obtained at pH 5.1-5.3. Mixed micelles associated with coupled colipase, but no modifications of lipase-colipase interactions took place when mixed micelles were added to the elution buffer. DMMA-modified coupled colipase failed to interact with lipase, owing to the specific orientation of the modified cofactor in the gel.     

Amino acid analysis by gas-liquid chromatography on a single column

A gas-liquid chromatography procedure for analysis of protein amino acids is described. Amino acids are esterified to their n-propyl esters then acylated to their heptafluorobutyryl (HFB) derivatives. These reactions were carried out in a single tube at 100°C. A simple steam-heating apparatus was constructed that heats only the bottom of the reaction vessel. Only 10 min were needed for esterification and 20 min for acylation, respectively. The resulting products, N-HFB-n-propyl esters of amino acids, were chromatographed on a single column. The amino acid compositions of chymotrypsinogen A and casein were analyzed by the present method, and the results were compared with those obtained by ion-exchange chromatography reported previously.     

Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.     

Amino acid analysis of culture media and autolysates by capillary gas chromatography

N-heptofluorbutyryl-O-propyl ethers from 20 natural amino acids were analysed on a capillary column Am-Ac by gas chromatography. A high reproducibility of the quantitative estimation of the compounds studied was achieved not only for the standard mixtures but when analysing the amino acid composition of the autolysates of the baker's yeast and cultural media.     

Bile acid kinetics in man studied by radio thin-layer chromatography and densitometry coupling

A method based on coupling of the techniques of radioscanning a TLC plate and densitometry has been developed for the determination of pool sizes and fractional turnover rate of bile acids in man after intraduodenal administration of ¹⁴C-labelled acid. The validity of the method has been checked by comparison of the results obtained with those of an enzymatic spectrophotometric analysis, and a measurement of the radioactivity by liquid scintillation counting, after elution of the separated bile acid from a TLC plate. Advantages of the proposed method over the previous one include a reduced number of manipulations, the possibility of automation, a better reproducibility, and the possibility of elaborating the radiometric data obtained for the primary bile acid for better characterising its metabolism inside the enterohepatic circulation.     

Protein-detergent interactions studied by capillary isotachophoresis

The interactions of sodium dodecyl sulphate with bovine serum albumin and ovalbumin have been studied by capillary isotachophoresis. This method makes it possible to determine very accurately the number of ligands bound to the high-affinity binding sites of the native protein. Bovine serum albumin was found to have seven high-affinity binding sites whereas ovalbumin in its native state was found to lack high-affinity binding sites for dodecyl sulphate.     

Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate

Methods for the quantitative derivatization of amino acids with phenylisothiocyanate and for the separation and quantitation of the resulting phenylthiocarbamyl derivatives by reverse-phase high-performance liquid chromatography are described. Phenylthiocarbamylation of amino acids proceeds smoothly in 5 to 10 min at room temperature. Coupling solvents, reagent, and some byproducts are removed by rotary evaporation under high vacuum, and the phenylthiocarbamyl derivatives are dissolved in 0.05 M ammonium acetate, pH 6.8, for injection onto the octyl or octadecylsilyl reverse-phase column. Columns are equilibrated with the same solvent and the effluent stream is monitored continuously at 254 nm for detection of the amino acid derivatives. Elution of all of the phenylthiocarbamyl amino acids is achieved in about 30 min utilizing gradients of increasing concentrations of ammonium acetate and acetonitrile or methanol. This approach to amino acid analysis offers select advantages, both with respect to methods which employ reverse-phase separation of prederivatized samples and to the classical ion-exchange procedure. All amino acids, including proline, are converted quantitatively to phenylthiocarbamyl compounds and these are stable enough to eliminate any need for in-line derivatization. Furthermore, results comparable in sensitivity and precision to those obtained by state-of-the-art ion-exchange analyzers may be generated with equipment that need not be dedicated to a single application.     

Amino acid/water interactions study: a new amino acid scale

Partition ratios of 8 free l-amino acids (Gln, Glu, His, Lys, Met, Ser, Thr, and Tyr) were measured in 10 different polymer/polymer aqueous two-phase systems containing 0.15?M NaCl in 0.01?M phosphate buffer, pH 7.4. The solute-specific coefficients representing the solute dipole/dipole, hydrogen-bonding and electrostatic interactions with the aqueous environment of the amino acids were determined by multiple linear regression analysis using a modified linear solvation energy relationship. The solute-specific coefficients determined in this study together with the solute-specific coefficients reported previously for amino acids with non-polar side-chains where used in a Quantitative Structure/Property Relationship analysis. It is shown that linear combinations of these solute-specific coefficients are correlated well with various physicochemical, structural, and biological properties of amino acids.     

Release of aminoacyl transfer ribonucleic acid from transfer ribonucleic acid synthetase studied by rapid molecular sieve chromatography

Complex of Ile-tRNA^Ile and isoleucyl-tRNA synthetase (IRS) was isolated by rapid chromatography on a Bio-Gel P-100 column at 4°C. By incubating the complex in the presence of excess unacylated tRNA^Ile prior to chromatography, it is possible to qualitatively measure the rate of exchange of Ile-tRNA^Ile with tRNA^Ile on the enzyme. The rate of exchange is markedly accelerated by isoleucine and isoleucinyl-AMP, but not by ATP. These results confirm previously published findings that the rate of release of newly synthesized Ile-tRNA^Ile from IRS is very slow in the absence of isoleucine or isoleucyl-AMP, but that the release is greatly enhanced by these ligands. The rapid chromatography procedure thus provides a very direct and straightforward means for measuring the dynamics of a proteinnucleic acid interaction.     

Amino acid conservation and interactions in rhodopsin: Probing receptor activation by NMR spectroscopy

Rhodopsin is a classical two-state G protein-coupled receptor (GPCR). In the dark, its 11-cis retinal chromophore serves as an inverse agonist to lock the receptor in an inactive state. Retinal–protein and protein–protein interactions have evolved to reduce the basal activity of the receptor in order to achieve low dark noise in the visual system. In contrast, absorption of light triggers rapid isomerization of the retinal, which drives the conversion of the receptor to a fully active conformation. Several specific protein–protein interactions have evolved that maintain the lifetime of the active state in order to increase the sensitivity of this receptor for dim-light vision in vertebrates. In this article, we review the molecular interactions that stabilize rhodopsin in the dark-state and describe the use of solid-state NMR spectroscopy for probing the structural changes that occur upon light-activation. Amino acid conservation provides a guide for those interactions that are common in the class A GPCRs as well as those that are unique to the visual system. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

Summary. Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).     

6-Aminohexanoic acid-plasminogen interactions studied by fluorescence

Circular polarization of luminescence spectra of human plasminogen and of its derivatives were measured in solutions of ligand-free proteins and with saturating amounts of 6-aminohexanoic acid. Spectroscopic changes induced by the ligand reveal similar perturbations of the binding sites in all the protein derivatives. It is concluded that the gross conformational change induced by 6-aminohexanoic acid binding to the native plasminogen involves changes of sterical relations of entire protein domains.     

Xylosylated naphthoic acid-amino acid conjugates for investigation of glycosaminoglycan priming

Three different series of xylosylated naphthoic acid-amino acid conjugates containing one or two amino acid residues were synthesized for the investigation of glycosaminoglycan priming and potential use as anti-tumor drugs. All xylosylated naphthoic acid-conjugates inhibited the growth of normal lung fibroblasts to some extent, whereas the growth of tumor derived T24 carcinoma cells was not affected. There was no correlation between amino acid conjugation, retention time and the antiproliferative activity. Only one compound initiated the priming of glycosaminoglycans. Modification of the naphthalene ring with one or two amino acid residues did not have any effect on proteoglycan biosynthesis or glycosaminoglycan priming in T24 carcinoma cells.     

Dendrimer-protein interactions studied by tryptophan room temperature phosphorescence

Dendrimers are a relatively new class of materials with unique molecular architectures, which provide promising opportunities for biological applications as DNA carriers and drug delivery systems. Progress in these fields, however, requires knowledge of their potential interactions with biological components at cellular and molecular level. This study utilizes Trp phosphorescence spectroscopy to examine possible perturbations of the protein native fold in solution by neutral, positively and negatively charged fifth generation polyamidoamine (PAMAM) dendrimers. Phosphorescence lifetime measurements, conducted on model proteins varying in the degree of burial of the triplet probe and in quaternary structure, show that dendrimers interact with proteins in solutions forming stable complexes in which the protein structure may be significantly altered, particularly in superficial, flexible regions of the polypeptide. Both electrostatic and non-electrostatic interactions can give rise to stable complexes, whose affinity and limited number of binding sites distinguish them from mere aspecific molecular associations. Of direct relevance for the application of these polymers in the medical field, structural alterations have also been detected in human plasma proteins such as serum albumin and immunoglobulins. The above results suggest that Trp phosphorescence may provide a useful monitor for working out experimental conditions and protocols that help preserve the structural integrity of proteins in the presence of these polymers.