Differential interferometry in the analytical ultracentrifuge. I. Design, construction, and alignment of differential interferometer

The application of an enzyme-linked immunosorbent assay to the study of histone antigens is described. This method is much more sensitive than microcomplement fixation tests and allows the interaction between histone antibodies and nucleosomes to be measured directly on the solid phase under a variety of ionic conditions. The results indicate that the enzyme-linked immunosorbent assay is a sensitive assay for investigating different conformational states of chromatin and for detecting low levels of histone antibodies in antisera.     

Rapid alignment of the interference mask in the ultracentrifuge

A procedure is given for measuring the degree of misalignment of the interference mask in the analytical ultracentrifuge. A simple apparatus is described which allows the mask alignment to be corrected by precise lateral movement of the mask.     

Use of the laryngeal mask airway in rabbits: placement and efficacy

The laryngeal mask airway (LMA) has been used in various animal species anesthetized for the purpose of device evaluation, but the device has not been evaluated in rabbits during surgery. The authors tested the feasibility and potential advantages of using the LMA in 50 rabbits undergoing surgery under spontaneous-breathing inhalational anesthesia, focusing mainly on the technique of insertion and its efficacy. The LMA was easily inserted and no air leakage at the larynx was detected. Although four rabbits developed lingual cyanosis, this was reversible and most likely due to lingual vascular compression by the LMA. The authors conclude that the LMA is an attractive alternative to endotracheal intubation, as the mask can be inserted easily and rapidly and its correct placement is easily confirmed.     

Application of the miniature ultracentrifuge in receptor-binding assays.

A new binding assay for membrane receptor systems has been developed employing an air-driven ultracentrifuge (Beckman Airfuge). The main advantages of this method for measurement of radioligand binding in aqueous medium are (i) the rapidity (30 s) in separating the bound from the unbound fraction, (ii) the small volume (100 μl) of assay medium which permits a relatively small excess of ligand over receptor to be employed, and (iii) the simplicity of manipulations which allows a high degree of replication. The variation in a triplicate set of assays is usually less than 0.5%. By virtue of maintaining equilibrium throughout the assay the present method is especially useful for ligands exhibiting rapid reversibility in binding. Binding of [³H]ouabain to several membrane (Na⁺, K⁺)-ATPases and binding of [³H]etorphine to the oplate receptor from brain membranes are discussed here. Also the inhibition of [³H]ouabain binding by Tris is discussed.     

Differential interferometry in the analytical ultracentrifuge. II. General applications

Various ways of applying differential interferometry to ultracentrifugal analyses are examined and several analytical techniques are established. In transport and moving boundary methods, the sedimentation coefficient is more precisely determined in the differential interference system than in the schlieren optical system because fringe measurement accuracy is much higher in the former system. Compared to interference and absorption optics, the differential interferometer provides a more exact s value in the transport method since an accurate calculation procedure can be adopted. Moreover, the following advantages of differential interferometry are noted. Determination of the initial solute concentration, which must be done in the usual interference method, is unnecessary in this sedimentation equilibrium method. Regardless of the partial loss of solute from the observed system due to rapid precipitation or adsorption to the cell wall during centrifugation, the molecular weight of the rest of the solute can be determined exactly. The diffusion coefficient can be determined accurately by fringe displacement analysis at the hinge point during the transient state. Together with the molecular weight and diffusion coefficient, the partial specific volume and sedimentation coefficient of a solute can be obtained from the result of a single low-speed centrifugation when the sample solutions in H₂O and D₂O are compared.     

Computer stimulation of sedimentation in the ultracentrifuge. VII. Solutes undergoing indefinite self-association

The velocity sedimentation of solutes involved in self-association equilibria of the indefinite type was simulated using a computer model. The changes in boundary shape that resulted from varying the association constant and the molecular weight of the self-associating monomer were examined. Both ideally and nonideally sedimenting solutes were considered, and several alternative treatments of the variation of the frictional ratio with molecular size were used. All of the calculated boundaries were skewed, with the leading limb of the gradient profile steeper than the trailing limb. For relatively tightly self-associating solutes, the boundaries were very broad and strongly skewed. No shoulders or subsidiary peaks were observed for any of the model solutes used.