Time-Resolved Analysis of Macromolecular Structures During Reactions by Stopped-Flow Electrooptics

Abstract

A stopped-flow field-jump instrument and its use for the analysis of macromolecular structure changes during reactions is described. The operation of the new instrument is simple and reliable, owing to a new type of cell construction with electrodes directly integrated in a quartz cuvette: major advantages are the relatively low demand on sample quantities and a high time resolution. The stopped flow is characterized by a dead time of ∼0.5 ms. Electric field pulses with field strengths up to 20 kV/cm and rise times in the nanosecond range are applied at adjustable times after stop of the flow. The time resolution of the optical detection is up to the nanosecond time range. The instrument may be used for the combination of stopped flow with temperature-jump and field-jump experiments. A particularly useful new application is the analysis of macromolecular reactions by electrooptical measurements, because electrooptical data provide information about structures. This is demonstrated for the intercalation of ethidium into double-helical DNA. The transients, measured at 313 nm, where the signal is exclusively due to ethidium bound to the DNA, demonstrate a relatively high negative dichroism at 0.5 ms after mixing. The absolute value of this negative dichroism increases in the millisecond time range and approaches the equilibrium value within about a second. The dichroism decay time constants demonstrate a clear increase of the effective DNA length due to ethidium binding, already 0.5 ms after mixing; a further increase to the equilibrium value is found in the millisecond time range. The analysis of these data demonstrate the existence of up to three relaxation processes, depending on the conditions of the experiments. The dichroism amplitudes, together with the decay time constants, indicate that all the reaction states found in the present investigation are complexes with insertion of ethidium residues between basepairs. Moreover, the data clearly show the degree of intercalation in the intermediate states, which is very useful information for the quantitative assignment of the mechanism.