Macro and micro rate zonal analytical centrifugation of polydisperse and slowly diffusing sedimenting systems in isovolumetric density gradients. Application to cartilage proteoglycans.

A method to study the polydispersity of zonally sedimenting and slowly diffusing macromolecules or particles in isokinetic or isovolumetric density gradients is presented. First, a brief theory is given for predicting the zonal profile after a "triangular" (or "inverse") zone is centrifuged. This type of zone is essential to preserve hydrodynamic stability of the very slowly diffusing polydisperse solutes. It is proven, both by semitheoretical considerations and by computer calculations, that the resulting concentration profile of macrosolute is almost identical with that obtainable with a rectangular zone coextensive with the triangular one and carrying the same total mass. Next, practical procedures are described for the convectionless layering of very small triangular zones (50 microL or less). The linearity and stability of the zones are experimentally tested and verified. Finally, the method is applied to cartilage proteoglycan preparations that included either the monomeric molecules only or both the monomeric and the aggregated ones. The zonal results are compared with those obtained by using conventional boundary sedimentation. The two sets of results are seen to coincide fairly well, thus proving that the present technique can add to preparative zonal centrifugation the analytical precision of boundary sedimentation. A multimodal polydisperse system is suggested to describe the aggregated proteoglycan macromolecules.

Optimization of the binding of dissociated exfoliated cervico-vaginal cells to glass microscope slides.

In order to monitor the development of a cell dissociation technique, it was essential to utilize the Centrifugal Cytology rotor to produce glutaraldehyde-fixed even cellular dispersions. The Cytology rotor has been improved to insure rapid alignment with the centrifugal field during both acceleration and deceleration, and the fixative is now delivered to the surface of the slide. The dissociation of the cells results in a loss of their adhesion to glass slides. Three bonding agents were tested: (a) Poly-L-Lysine; (b) Mayer's albumin fixative; (c) positively charging the slides with a silicone coating. The results with 65% albumin-coated slides were clearly superior to the other two. The addition of a postfixation step of 95% ethanol/4% polyethylene glycol did not significantly affect the recovery of the cells, but did eliminate some unevenness in the Centrifugal Cytology preparations, flattened the cells and expedited the procedure.

Biohazards assessment in large-scale zonal centrifugation.

A study was conducted to determine the biohazards associated with use of the large-scale zonal centrifuge for purification of moderate risk oncogenic viruses. To safely and conveniently assess the hazard, coliphage T3 was substituted for the virus in a typical processing procedure performed in a National Cancer Institute contract laboratory. Risk of personnel exposure was found to be minimal during optimal operation but definite potential for virus release from a number of centrifuge components during mechanical malfunction was shown by assay of surface, liquid, and air samples collected during the processing. High concentration of phage was detected in the turbine air exhaust and the seal coolant system when faulty seals were employed. The simulant virus was also found on both the centrifuge chamber interior and rotor surfaces.

Large-volume purification of tumor viruses by use of zonal centrifuges.

Large volumes of fluids from selected cell cultures producing ribonucleic acid tumor viruses were processed by a double sucrose density gradient procedure using zonal centrifuges. The primary recovery utilized a Model K continuous-flow zonal centrifuge at 9 to 11 liters/hr. The virus zone from the Model K gradient was further purified on a second gradient in a B-29 rotor. The resulting viral concentrates at 2 x 10(11) particles per ml showed high purity by electron microscopy and gel analysis and were useful reagents in biochemical and immunological studies of the reverse transcriptase enzyme, virus structure, complement fixation, and other virus properties. Similar recovery methods were applied to other tumor virus systems.

Respiratory syncytial virus isolation by combined continuous flow-isopycnic banding centrifugation.

A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition.