O. Kratky

Studies on the structure of lipoprotein A of human high density lipoprotein HDL3: the spherically averaged electron density distribution.

1. Introduction In a previous article [l] we gave a first report on the experimental results obtained by X-ray small angle scattering of the LpA fraction of human plasma high density lipoprotein HDL3 in 0.15 M NaCl solution. The shape of the scattering curve showing three characteristic side maxima reflected a high structural regularity of the molecule. In the present paper we give a more detailed analysis of the scattering curve mentioned above, as well as additional results from X-ray small angle scattering experiments in 36% sucrose solution. The results are strongly in favour of a spherical molecular model of 96 A diameter consisting of an inner part of low elec- tron density surrounded by a high electron density shell.

X-ray small angle scattering of human plasma high density lipoprotein LpA from HDL2: application of a new evaluation method.

In their recent study, Shipley et al. [ 1 ] presented data on the structure of HDL, as obtained from X-ray small angle scattering. The results were in good qualitative agreement with the results of our previous studies on LpA from HDL3 [2,3] indicating a common molecular architecture for these two species of human plasma high density lipoproteins. However, the proposed model for HDL, was based only on comparison of theoretical and experimental scattering curves, obtained from experiments in solvents of one single electron density. In this case where a non homogeneous electron density within the molecule must be assumed a priori, the fit of the data is only to be considered as a necessary but not sufficient condition. Therefore we extended our studies on lipoproteins also to the HDL, species. This article is a report on the results which were obtained from X-ray small angle scattering of LpA from HDL, in different solvents. For the translation of the scattering data into real space information we employed for the first time a novel procedure

Small angle X-ray scattering of LpA, the major lipoprotein family of human plasma high density lipoprotein HDL3

Most of the present knowledge of size and shape of human plasma high density lipoproteins (HDL) is based on their visualization by electron microscopy using negative staining techniques [ 1,2]. These experiments indicate a subunit structure for both the classes of high density lipoproteins, HDb and HDL3. Unfortunately the resolution has been limited to about 30 A, which did not allow to give precise dimensions of the subunit structure. Moreover, these pictures revealed a great variety of possible arrangements for the subunits within the particles of each class. The assumption of a subunit structure of the constituent protein moiety was strongly supported by delipidation experiments. Several groups of investigators have shown [3-51, that the apoprotein can be fractionated into nonidentical polypeptides. It is a well established fact, that lipoprotein density classes are heterogeneous with respect to their chemical, immunochemical and physicbchemical properties [6-81. In fact there does not seem to exist any density fraction, from which a single, homogeneous lipoprotein fraction can be isolated solely by ultracentrifugation. We therefore applied additional purification procedures to isolate a lipoprotein A (LpA) preparation from HDLJ fraction of human serum, homogeneous with respect to its immunochemical and physicochemical behaviour. This paper is a first report of our X-ray small angle scattering experiments on LpA. The results given are confined to the most unambiguous molecular parameters as the radius of gyration, the molecular weight and the particle volume. The results indicate a subunit structure for LpA.

Röntgenkleinwinkeluntersuchungen an Lösungen eines Polystyrolpräparates mit enger Molekulargewichtsverteilung

ZusammenfassungEs wurde das Streuverhalten von Polystyrol im thermodynamisch „schlechten” Lösungsmittel Cyclohexan zwischen Entmischungstemperatur undϑ-Temperatur untersucht. Beim ϑ-Punkt und nahe dabei liegt das Verhalten eines statistisch verknäuelten Fadenmoleküls vor. Unterhalb derϑ-Temperatur beginnt eine Aggregation. Knapp oberhalb der Entmischungstemperatur entspricht das Molekulargewicht etwa einer Verhängung zweier Fadenmoleküle. Ein Auftreten von kompakten Polymerknäueln ist bei dieser Temperatur nicht festzustellen. Der Einfluß des Fadenquerschnitts auf die Streukurve ist beträchtlich, da der Querschnitt nicht viel kleiner als die Persistenzlänge ist. Trotzdem läßt sich die Persistenzlänge (a=12,2 Å) aus dem Übergangspunkt zwischen den etwa 1/(2ϑ)2 und 1/(2ϑ) proportionalen Bereichen bestimmen, da der Knick im Übergangsbereich infolge der großen Anzahl von Persistenzlängen pro Molekül sehr ausgeprägt ist.AbstractInvestigations were made of the scattering behaviour of polystyrene in the thermodynamically “bad” solvent cyclohexane between precipitation-temperature andϑ-temperature. At theϑ-point and close to it the behaviour of a statistically coiled chain molecule is found. Belowϑ-temperature an aggregation is beginning. Just above precipitation-temperature the molecular weight corresponds to an entanglement of two chain molecules. Appearance of compact polymer coils could not be noted at this temperature. The influence of the chain crosssection upon the scattering curve is considerable as the crosssection is not much smaller than the persistence length. Nevertheless, the persistence length (a=12.2 Å) may be determined from the transition point between the ranges roughly proportional to 1/(2ϑ)2 and 1/(2ϑ) as the break within the transition range is very distinct on account of the great number of persistence lengths per molecule.

X-ray small-angle scattering on soluble antigen—antibody complexes

By ultracentrifugal and electrophoretical studies Singer and Campbell [l] have provided data on the composition and conformation of soluble antigenantibody complexes formed in excess antigen. They found that with a large antigen excess, a complex ‘A complex’ is formed composed of two antigens bound by one antibody molecule (Ag, Ab) which are arranged in a linear array. Their findings concerning the composition of the A complex were confirmed by a number of investigations [2, 31. Electron microscopic studies by Feinstein and Rowe [4] and Valentine [5] have shown DNP labelled ferritin molecules bridged by anti-DNP antibodies, the latter having a more or less flexible Yshaped structure. Unfortunately the distances between the ferritin molecules varied within too large a range for precise dimensions of the complexes to be given. All electron micrographs revealed markedly smaller dimensions for the complexes than expected from the hydrodynamic [6] and X-ray data [7] available for yG immunoglobulin. These discrepancies might be ascribed partially to the difficulties in the staining method [8] and to total dehydration in the vacuum necessary for electron microscopy. However it is now well established that the antigens within the A complex are bound on the extreme ends of the respective F(ab) parts of the antibody molecule. The present paper is a report of our attempts to obtain additional data on the conformation of the A complex with the aid of the small angle X-ray scattering method, which in the past has contributed information on the structure of immunoglobulin [7,9, lo] , The work

Röntgenkleinwinkeluntersuchungen an niedermolekularen Cellulosenitraten

SummaryLow molecular cellulose nitrates dissolved in acetone and in a poor solvent are investigated by X-ray smallangle scattering up to aBragg value of 800 Å. As reported in an earlier paper there exists a clear dependence of the particle weight and the radius of gyration of dissolved cellulose nitrates on the solvent used and on the degree of ageing of the samples. This paper gives further experimental evidence of this fact. Moreover ageing causes an increase of the persistence length which is shown on two samples investigated both in fresh and aged state. This result is discussed on the basis of partial association of fully denitrated ranges of the chains.

Evidence for the absence of a central core in particles aggregated from protein subunits of bacteriophage fr

A method has been described for the formation of virus-like protein particles from isolated protein subunits of the icosahedral bacteriophage fr [I] . This demonstrated for the first time that self-assembly is possible for the coat protein of icosahedral viruses in the absence of nucleic acid. The structure of these virus-like particles is still controversial. It was first assumed that they were empty shells corresponding to the protein shell of the virus [l] . This was supported by electron microscopic observations [2-41 . On the contrary, a model for these particles was derived from sedimentation analysis and also from electron microscopic observations which showed a viruslike shell surrounding a central protein core. This core was thought to consist of 60 subunits [5,6]. It seems that the latter model has been widely accepted [7-91. Since small angle X-ray scattering should be particularly capable of clearing up these conflicting results and hypotheses, we have performed a study on the reaggregated particles by this technique. By comparison of the scattering curves for these particles with those for empty protein shells derived from the phage by alkaline degradation of the RNA [IO] and with theoretical scattering curves for the different models, we are now able to show that the reaggregated virus-like particles are indeed empty shells which are very similar to the protein shell of the virus.

Röntgenkleinwinkeluntersuchungen an Lösungen von Schweineserum-Albumin bei verschiedenen pH-Werten

Higly purified samples of porcine serum albumin were measured by the X-ray small-angle scattering method at pH 7.0, 5.1, and 3.7. The molecular weight is found to be the same in all three cases: the mean value is 70 300±2 500. The radius of gyration is 31.1 Å and 33.0 Å at pH 5.1 and 7.0 resp., whereas at pH 3.7 a value of 37.8 Å is found. The determination of the molecular conformation yields a similar axial ratio at pH 5.1 and pH 7.0 of 0.75 : 1 : 2; at pH 3,7 we find an axial ratio of 0.2 : 1 : 1 to be consistent with the scattering curves.ZusammenfassungSchweineserum-Albumin wurde nach sorgfältiger Reinigung bei den pH-Werten 7,0, 5,1 und 3,7 mit Hilfe der Röntgenkleinwinkelstreuung untersucht. Es wurde bei allen Messungen praktisch dasselbe Molekulargewicht gefunden: der Mittelwert liegt bei 70 300±2 500. Die Ermittlung der Streumassenradien ergab bei pH 7,0 und pH 5,1 ähnliche Werte (33,0 Å bzw. 31,1 Å), bei pH 3,7 dagegen einen Wert von 37,8 Å. Die Formbestimmung ergab bei pH 7,0 und pH 5,1 identische Achsenverhältnisse (0,75 : 1 : 2), während bei pH 3,7 ein solches von 0,2 : 1 : 1 gefunden wurde.