ULTRACENTRIFUGING

From Big Medical Encyclopedia

At LTRATsENTRIFUGYROVANIE — a method of division of hl. obr. high-molecular compounds, their units, subcellular structures and viruses under the influence of centrifugal force. Generally At. use in the scientific researcher - skoy to work. However analytical At. it can effectively be applied to statement and specification of the diagnosis, napr, at quantitative definition of lipoproteids (see) blood sera for the purpose of establishment like lipoproteinemiya, to the analysis of immunoglobulins (see) blood sera, identification of viruses (see), etc.

At. make in analytical or preparative ultracentrifuges, the speed of rotation of a rotor in to-rykh reaches 80 000 turns in min., and acceleration of centrifugal force in hundreds of thousands of times exceeds acceleration of force of terrestrial inclination. In such conditions there is a sedimentation (see) not only a coarse break-up, but also the dissolved macromolecules — proteins (see), nucleic acids (see), polysaccharides (see) etc.

The rotor of the ultracentrifuge rotates in vacuum with a pressure apprx. 10-3.shyurt. the Art., created forvacuum and diffusion pumps, is also set in motion most often by electric motors. In analytical ultracentrifuges insert the so-called cells representing metal cylinders with the central insert having a sectorial cavity and clamped between two quartz glasses into openings of a rotor. The volume of this cavity makes 1 ml and less. The volume of test tubes in preparative ultracentrifuges depends on the maximum speed of rotation of a rotor.

For overseeing several optical systems are available process of sedimentation and registration of borders of sedimentation in analytical ultracentrifuges, from to-rykh most often use absorbing and refraktometrichesky (so-called went - ren-system). The absorbing system registers attenuation range of light solution in a cell depending on distance from an axis of rotation. This system is applied at a research of the diluted solutions (concentration apprx. 0,005%) nucleinic to - t (at the wavelength of 260 nanometers) and proteins (at the wavelength of 230 nanometers). Went - the ren-system allows to register gradients of indices of refraction (or concentration) in the form of the peaks corresponding to borders of sedimentation or flotation. Shliren-sistemu apply to the analysis of solutions of proteins and other high-molecular compounds with concentration apprx. 0,5%.

By the main methods analytical At. determination of speed of sedimentation and sedimentation equilibrium are. In the first case it is about the movement of border of sedimentation from a meniscus to a bottom of a cell or border of flotation from a bottom of a cell to a meniscus. This method is applied to definition of homogeneity of substances, their coefficients of sedimentation or flotation, and also to determination of other parameters (see Sedimentation). During the definition of sedimentation equilibrium at At. such state when the flow of substance due to sedimentation is compared to a flow due to diffusion is reached. This method use a pier for definition. weight (weight).

By the main methods preparative At. are differential, zone and high-speed and equilibrium (isopycnic) by U. Differentsialnoye U. it is based on separate sedimentation of particles on a bottom of test tubes. For this purpose select such speed and time of rotation of a rotor of the ultracentrifuge that in draft there were most coarse particles, and in nadosadochny liquid there were less large. During the ultracentrifuging of 1 g of homogenate of fabric (after its upholding within

20 min.) at a speed of rotation of a rotor of the ultracentrifuge corresponding to acceleration of centrifugal force of 1000 g in 5 — 20 min. on a bottom of a centrifugal test tube settle kernels and remained not destroyed (intact) cells, at 10 000 g (20 min.) — mitochondrions, lysosomes, etc., at 100 000 g (1 — 2 hour) — free ribosomes and microsomes, at further centrifuging with a speed of rotation of a rotor corresponding to acceleration about 400 000 g are besieged the dissolved proteins, nucleinic to - you and other macromolecules. At zone and high-speed At. solution of razdelyaekhmy components is layered on more dense environment. The last is trained, creating in a test tube for At. a gradient of concentration of sucrose (apply also glycerin, fikoll, etc.), napr, from 10% at a meniscus to 30% at a bottom. Such gradient of concentration prevents convection washing out of zones of the sedimentating components. After achievement of optimum division of zones U. stop, take zones (e.g., puncturing a bottom of a test tube with a needle from the syringe, i.e. «digging out a gradient»). Then study components of the allocated zones on these or those properties. In this case division of proteins, nucleinic to - t, etc. happens according to their coefficients of sedimentation. At equilibrium (isopycnic) At. test tubes fill with solution of the divided components in the presence of large numbers, naira., chloride CsCl2 caesium (apply also sulfate Cs2S04 caesium, metrizamid, etc.). In time At. there is a gradient of density of chloride caesium, and the meniscus shall have density of solution less, and density of solution at a bottom — is more than density of the studied components. In that case these components will sedimentate or float until reach a layer, in Krom density of chloride caesium is equal to the floating density of the studied components (so-called isopycnic area). At the same time the movement of components will stop and there will be an equilibrium state. Sampling is made as well as at zone and high-speed At. Unmixing of particles at equilibrium At. it is carried out not on coefficients of sedimentation, and on floating density.

For preparative At. apply rotors of different types. In angular rotors of a test tube are inserted into nests at an angle to an axis of rotation, sedimentation in this case happens in the direction of radius on walls of test tubes, and on them — on a bottom of test tubes. These rotors usually apply to differential U. V rotors with freely suspended sleeves with test tubes (so-called baket-rotors) of a sleeve during rotation of a rotor horizontal position and sedimentation accept occurs parallel to walls of test tubes. Such rotors usually use for zone and equilibrium At. with gradients of concentration or density of supporting medium. For the same purposes began to use so-called zone rotors. These rotors have big shank bore, in a cut creation of a gradient (e.g., concentration of sucrose) and sampling happens at small speeds of rotation of a rotor (apprx. 2000 — 3000 turns in min.), and division is carried out at the maximum speeds of its rotation.

Bibliography: Bowen T. Introduction to ultracentrifuging, the lane with English, M., 1973; Osterman L. A. Methods of a research of proteins and nucleic acids, Electrophoresis and ultracentrifuging, M., 1981. V. O. Shpikiter.

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