Richard D. Spencer

Influence of Brownian Rotations and Energy Transfer upon the Measurements of Fluorescence Lifetime

The depolarization of the fluorescence of solutions by either Brownian rotations or intermolecular energy transfer may be simply described by a system of first‐order linear differential equations containing as only parameters the rate of fluorescence emission and the rate of transport of the excitation from one orthogonal component of the emission to another. The steady‐state solution has the form of Perrins equation describing the depolarization by Brownian rotations, and the time‐dependent depolarization following a unit light impulse is that originally described by Jablonski. The solution for sinusoidal excitation is novel in that: 1. It shows the difference in lifetime between the polarized components of the emission to be a sensitive function of the ratio of the modulation frequency ω to the emission rate λ. For ω/λ > 1 the difference between the polarized lifetimes may become many times greater than that observed after a unit light impulse. 2. It permits the determination of both the rate of transp...

Fluorescence polarization: measurements with a photon-counting photometer.

A T-format, photon-counting polarization photometer was constructed to facilitate precise polarization measurements at low signal-to-noise ratios. The instrument optics and electronics are described. Several examples of the instruments performance are given, including measurements on picomolar fluorescein solutions at low excitation resolution and the excitation polarization spectrum of indole (2 x 10(-4)M) at a resolution of 5 A.

Construction and performance of a scanning, photon‐counting spectrofluorometer

A photon‐counting spectrofluorometer interfaced to a Nuclear Data ND 812 computer was constructed to facilitate acquisition of emission spectra at very low signal‐to‐noise ratios. The photon‐counting technique combined with repetitive scanning permits the detection and quantitation of emission from strong fluorophors near the picomolar level. The computer performs a variety of digital manipulations such as subtraction of background, normalization of spectra, corrections for instrument response, and polarization calculations. Special attention needs to be given to handling of cuvets and solvents to minimize background signals.

Direct fluorimetric assay for proteolytic activity against intact proteins

Abstract The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates resulting in a decline in fluorescence. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates in this assay procedure. The activity of 1 μg chymotrypsin or trypsin and 100 ng pepsin could be easily detected by this method within 4 to 8 min depending upon the protein substrate. The digestive enzymes and bromelain exhibited activity against most if not all six of the protein substrates used. In contrast, the extract from germinating pumpkin seeds exhibited significant activity only against pumpkin seed globulin, with maximal activity at pH 7.4. Compared with the assay method for proteolytic activity utilizing ninhydrin analysis of the reaction products, this method was at least 10 times more rapid and gave significant detectable activity with much lower quantities of proteolytic enzyme.