Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation.

The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3S subunit forming a 7S intermediate. At a level of 75-80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place. The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 x 10(-7) cm2 . s-1 a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.

Isolation of the 12 S globulin from rapeseed (Brassica napus L.) and characterization as a "neutral" protein. On seed proteins. Part 13.

An isolation procedure for the 12 S rapeseed globulin is described which includes precipitation by dialysis, purification using gel chromatography on Sephadex G-200, and ion-exchange chromatography on DEAE-Sephadex A-50. The isolated globulin represents a neutral protein with an isoelectric point at pH 7.25--determined by isoelectric focusing--and a relation of the acidic to basic amino acid residues (epsilon Glu, Asp--Amide ammonia: epsilon Arg, Lys, His) of 1.0. As in other storage globulins high contents of glutamic (19%) and aspartic (10%) acid and a low content of sulphur containing amino acids are characteristic for the amino acid composition. Amongst the basic amino acids arginine has the highest percentage (7%). Opposite to results of other authors the sugar content of the globulin is low (0.5%). From the amino acid composition an average hydrophobicity according to Bigelow was calculated which amounts to be 1041 cal/res. (4.36 kJ/res.).

[Chemical modification of proteins. 7. Effect of succinylation on some physico-chemical and functional properties of casein]. / Chemische Modifizierung von Proteinen 7. Mitt. Beeinflussung physikochemischer und funktioneller Eigenschaften von Casein durch Succinylierung.

Succinylation of amino groups of the casein complex results in a spontaneous dissociation of the protein into low-molecular 1.8-S components. Succinylated samples of casein are characterized by a higher electrophoretic mobility of the components in alkaline mediums and by a lower mobility in acidic buffer systems. The anodic migration in alkaline buffer systems increases depending on the degree of modification. This effect is more pronounced for the beta-casein than for the alpha s-casein fraction. In the potentiometric acid-base titration of the modified casein a hysteresis occurs during the back-titration. Increasing with the degree of modification, this effect points to a more or less strong break-up of the structure (entropy production). The solubility minimum of casein is shifted to lower pH values by succinylation. Samples succinylated to 90 or more per cent of the amino groups are insoluble at pH less than 3.5. Succinylated casein shows at a moderate degree of substitution (approximately equal to 40%) a highly increased water adsorption capacity. The oil adsorption capacity of the protein decreases after succinylation. Amongst the modified casein samples those ones with the highest water adsorption show the highest oil adsorption, too. The emulsifying capacity of low (approximately equal to 20%) succinylated casein samples decreases below the value determined for the unmodified protein. At higher modified samples the emulsifying capacity increases as the degree of succinylation increases. The emulsifying activity and emulsion stability of modified samples are lower than that measured for the parent protein. The foaming capacity of the casein is not improved by succinylation.

Hydrodynamic and quasi-elastic light scattering studies on the 12S globulin from rapeseed.

The 12S globulin, one of the major storage proteins of rapeseeds, has the following physico-chemical constants, as determined by ultracentrifugation, quasi-elastic light scattering measurements and gel chromatography: sedimentation coefficient S20(0), w = 12.7 x 10(-13) s; diffusion coefficient (quasi-elastic light scattering) D20(0), w = 3.8 x 10(-7) cm2 S-1; Stokes radius (by quasi-elastic light scattering) Rs = 5.7 nm and (by gel chromatography) Rs = 5.5 nm; partial specific volume (calculated from the amino acid composition) v(-) = 0.729 ml g-1; molecular weight Ms, D = 300,000 daltons, Ms, Rs = 294,000 daltons (Rs from the gel chromatography); frictional ratio f/fo = 1.28.

[Chemical modification of proteins. 5. Modification of the 11-S-globulin from sunflower seed by reaction with dialdehyde starch]. / Chemische Modifizierung von Proteinen. 5. Mitt. Modifizierung der 11-S-Globulins aus Sonnenblumensamen durch Reaktion mit Dialdehydstärke.

Dialdehyde starch (DAS) reacts unspecifically with the amino acid residues of the 11 S globulin from sunflower seed. The modification of the protein causes a decrease of the content of each amino acid. Their blocking reaches maximum values at high pH levels (9,5) and high concentration of protein (5%). Especially high reactivity is shown by arginine as well as by the hydrophobic amino acids isoleucine, valine, and proline, and furthermore by histidine, lysine, asparagine (aspartic acid), and glutamine (glutamic acid). By reaction with DAS at pH 8.0 70% of the amino groups are blocked within 6 h; on the contrary, glyoxale blocks only 30% of the amino groups. Owing to the blockage of charged amino acid groups, a shift of the isoelectric point of the protein to a lower pH (4,3-4,4) takes place; this effect can be followed for 2 days. As a result of the reaction with DAS, only small amounts (10-15%) of intermolecular crosslinkage products with sedimentation coefficients of 17 S and greater than 17 S were formed. But by means of SDS-gel electrophoresis, dimers and trimers of the polypeptide chains in the protein were detected.

[Isolation and characterization of 11-S-globulins from sunflower seeds (Helianthus annuus L.)]. / Isolierung und Charakterisierung des 11-S-Globulins aus Sonnenblumensamen (Helianthus annuus L.)

The isolation of II-S globulin, which is homogenous on sedimentation and gel chromatography (s020, w = II,8 S), from sunflower seeds is achieved by cryoprecipitation from solutions of high ion strength (I approximately 0.5 to 1.8) with simultaneous reduction of the salt concentration (I approximately 0.3) and repeated gel chromatography on Sephadex G-200. By this technique, the 7-S component which occurs in small quantity is isolated too. In solutions of low ion strength (I less than 0,3), the II-S globulin dissociates to give a 7-S component. At high or low pH values (greater than 9.0 and less than 3.0, respectively), the latter dissociates to give a 3-S component, and in the presence of urea it further dissociates to give subunits. In the presence of sodium of sodium chloride, the dissociation of the 3-S component may be inhibited. The various steps of dissociation can be studied by means of ultracentrifugal sedimentation, gel chromatography and gel electrophoresis.