David A. Yphantis

Fluorescent monitoring of SDS gel electrophoresis.

Abstract Reaction of proteins with dansyl chloride in mildly alkaline SDS systems at high mole ratios of dansyl chloride to protein yields dansylated proteins that can be run in SDS gel electrophoresis systems with no significant change in protein mobility. The procedure is simple

Improved ultracentrifuge cells for high-speed sedimentation equilibrium studies with interference optics

Abstract A new type of three-channel ultracentrifuge cell is deseribed; it is convenient and reliable in use for high-speed and long-term sedimentation equilibrium experiments employing the Rayleigh interference optical system. The plastic centerpiece of the cell has six compartments, each of which opens to the side through two filling holes that permit the chambers to be loaded and cleaned without disassembly of the cell. The nature of optical aberrations often seen with conventional cell assemblies at high rotational speeds has been analyzed and ways to avoid these difficulties are suggested. When the new cells are used according to a recommended routine and with improved components that minimize window strain, changes in blank patterns after long operation at speeds above 50,000 rpm remain within the standard error of reading interference plates. The increased optical reliability of the side-access cells helps to extend the routine application of meniscus depletion type molecular weight determinations to include molecules with sizes around 10,000 daltons.

Oligomerization of a MutS Mismatch Repair Protein from Thermus aquaticus

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization ofTaq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 μm. The ΔG 0 2–4 for the dimer to tetramer transition is approximately −6.9 ± 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

Existence and inhibition of hydrolytic enzymes attacking paramyosin in myofibrillar extracts of Mercenaria,mercenaria

Paramyosin is attacked by at least two hydrolytic enzymes in extracts of the adductor muscle of M.,mercenaria under the usual isolation conditions. KDTA inhibited and phenylmethanesulfonyl fluoride inhibited hydrolase activities have been identified. When paramyosin is extracted into high salt at pH 7.5 in the presence of 0.01M EDTA, we isolate a new molecular species of paramyosin with a molecular weight ∼5,000 daltons higher than material isolated by previous procedures. A high resolution acrylamide gel electrophoresis technique employing split gels was used to detect small differences in apparent sub-unit molecular weight.

Pulsed laser interferometry (PLI) in the analytical ultracentrifuge: I. System design

Abstract A description is given of a pulsed argon ion laser light source that is synchronized with rotor orientation in the analytical ultracentrifuge. The system described provides improved fringe contrast and resolution. In addition the light intensity passed by the optical system is several orders of magnitude greater than the usual high-resolution Rayleigh system so that either exposure times may be reduced to single flashes of 6 μsec on the usual photographic plates or, alternatively, slower emulsions of much higher resolution may be utilized in a practical manner.

RAPID PRECISION INTERFEROMETRY FOR THE ANALYTICAL ULTRACENTRIFUGE

This is the first of a series of manuscripts presenting methods to enable rapid reduction of data from the Rayleigh interference optical system of the Beckman Model E analytical ultracentrifuge. Here we present a pulsed laser controller for the ultracentrifuge. This laser controller uses a phase-lock-loop to provide properly timed light pulses over the speed range of 3000 to 60,000 rpm; it effectively resolves one rotor revolution into 4096 discrete angular positions. The circuit has been designed so that the laser light bursts occur at selectable angular positions of the rotor that are independent of rotor speed even under conditions of maximum acceleration or deceleration. We have used this controller in our laboratory over a 7-year period for both photographic and real-time collection at interferometric data from the ultracentrifuge.

Rapid precision interferometry for the analytical ultracentrifuge: III. Determination of period of rotation, frequency of rotation, and elapsed time

The approaches presented in this series of papers make possible rapid gathering, reduction, and analysis of data from the Rayleigh interference optical system of an analytical ultracentrifuge. Instrumentation described in this paper provides some of the timing and measurement circuits necessary for a microprocessor or minicomputer to determine the rotor frequency, rotor period, and elapsed time of an experiment. It includes simple but effective circuits to generate precise rotor timing pulses that are useful for synchronization of pulsed light sources. Circuits to control photographic operations in the ultracentrifuge are described briefly. All of these circuits are interfaced to a simple microcomputer address/data bus. An adapter between this bus and a Q-bus (for a DEC LSI-11/2 or LSI 11/23 microcomputer) is also described. The circuits presented have been used in this laboratory over a 3-year period. They have proven reliable and form an integral part of the real-time data acquisition systems that have been constructed.

Rectangular Approximation for Concentration‐Dependent Sedimentation in the Ultracentrifuge

We derive an exact solution to the rectangular approximation of the Lamm equation where s=s0(1—kc). Properties of the solution are examined for approach to equilibrium and for velocity sedimentation.

Numerical study of the Johnston-Ogston effect in two-component systems.

Numerical solutions of the Lamm equation are presented for systems exhibiting the Johnston--Ogston effect. From these solutions it is apparent that the movement of the maxima of the concentration gradient curves reflects the sedimentation velocity of the slow or fast components in their appropriate plateaus. A simple generalization of the Johnston--Ogston analysis is presented, valid for all centrifugation times in a radial field and sector shaped cell provided only that there exist both a plateau of the slow component by itself and the mixed plateau with both slow and fast components present.

On Line Data Acquisition for the Rayleigh Interference Optical System of the Analytical Ultracentrifuge

The fundamental measurement in analytical ultracentrifugation is the concentration as a function of radial position. The Rayleigh interferometer of the analytical ultracentrifuge produces a cell image in which the concentration at each radial position is presented as the vertical displacement of a set of equally-spaced horizontal fringes (Richards and Schachman, 1959). Manual acquisition of data from interferograms is tedious and automated photographic plate readers still require that photographs be taken, processed, aligned and read before data analysis can be performed. Fortunately, a Rayleigh interference image is well suited for television- camera-based data acquisition. Described here are two types of automated Rayleigh interferometers for the Beckman Model E analytical ultracentrifuge. One type of system relays and magnifies the Rayleigh interference image from the usual photographic plane to a television camera located behind this plane. The other system uses a redesigned camera- cylinder lens combination to create a radially demagnified Rayleigh interference image of the cell on the television camera sensor located on the original cylinder lens mount.