James A. Knopp

Intracellular measurement of oxygen by quenching of fluorescence of pyrenebutyric acid.

Abstract We have examined pyrenebutyric acid as a possible fluorescent probe to determine intracellular concentrations of oxygen. Isolated rat liver cells rapidly take up pyrenebutyric acid, and the fluorescence of the cells is quenched by oxygen to the same extent as is free pyrenebutyric acid. Therefore, this technique appears to be applicable to the physiological range of oxygen concentrations.

Intracellular oxygen measurements of mouse liver cells using quantitative fluorescence video microscopy

Our currently developed fluorescence video microscope can measure fluorescence intensities with an error of +/- 1.5% of full scale in 65536 different positions of a microscope field. With a video frame freeze acquisition time of 33 ms, time-dependent changes of this order of time or slower can be followed. Using cells which have absorbed pyrene-1-butyrate to an intracellular concentration of 0.05 to 1 mM, the changes in fluorescence intensity with oxygen concentration are easily measured. The spatial resolution for data collection is 0.5 micron when a 54X objective is used. The individual Stern-Volmer quenching constants of each individual pixel were measured for agar slices and mouse liver cells treated with pyrenebutyric acid. The distribution of quenching constants for agar follows a normal curve about a mean value of 16 . 10(-4) torr-1. The data for mouse liver cells gave a non-normal distribution of quenching constants with a mean value of 18 . 10(-4) torr-1. The greater spread of the data from cells is interpreted as evidence for a real biological variation in the solubility coefficient of oxygen in different locations within the cell. In all the cells examined, this distribution has been observed to be non-random and appears to be associated with specific cell structures.

EFFECT OF TISSUE ABSORPTION AND MICROSCOPE OPTICAL PARAMETERS ON THE DEPTH OF PENETRATION FOR FLUORESCENCE AND REFLECTANCE MEASUREMENTS OF TISSUE SAMPLES

Abstract— An equation has been derived that is concerned with the measurements of fluorescence or reflectance intensities in reflectance or incident light microscopy. A theoretical basis for the linear relationship between relative fluorescence and reflectance changes is given. This equation predicts both the contributions of different volume elements below the surface of a tissue sample to the total intensity and the depth of penetration of the measured intensities. The relationship requires knowledge of the effective focal points of the excitation beam and the apparent absorption coefficient of the sample. Two procedures are given to provide the empirical values of the effective focal point. The absorption coefficient of mouse liver tissue at 420 nm was determined to be 3.3/mm. The fluorescence intensities of liver slices of differing thickness containing pyrene butyric acid were measured and were found to compare favorably with the calculated values for tissue absorption of 3/mm.

The binding of 8-anilino-1-naphthalene sulfonate (ANS) to fish myosin and the effect of salts on the thermal transitions of fish myosin-ANS complex

Myosin prepared from tilapia (Serotherodon aureus) was complexed with 8-anilino-1-naphthalene sulfonate (ANS) and continuously heated at 1 degree C/min. A large increase in fluorescence was observed with a transition temperature of 34 degrees C. The effect of several salts on the transition temperature was tested. A plot based on the equation of E. E. Schrier and E. B. Schrier [(1967) J. Phys. Chem. 71, 1851-1860] gave a value of less than or equal to 500 cal/mol-deg for the change in enthalpy per residue due to exposure to solvent. The ratio of hydrophobic group to amide group exposure to solvent was intermediate compared with the ratio of RNase and gelatin. Fluorescence titrations yielded one high affinity site with a Kb of 2 X 10(6) M-1 and at least 200 low affinity sites with an average value of 1 X 10(5) M-1. The parameters did not change significantly with temperature. We propose that the increase in ANS fluorescence reflects changes in conformation of myosin as monitored by these low affinity sites, resulting in an increase in surface hydrophobicity and representing a small enthalpic change in the conformation of the myosin molecule. As a consequence, the change in conformation accelerates polymerization of myosin oligomers.

CEREBRAL MICROCIRCULATORY CHANGES DURING EXPOSURE TO HYPOXIA

Changes in the red blood cell content of the cat cerebral cortical vessels were monitored using reflecting light during a decrease in PIO2. The following observations were made: 1) the capillary bed red blood cell content increased during hypoxia prior to the arteriole response at an arterial oxygen tension of 52.0 mmHg; 2) at arterial oxygen tensions below 52 mmHg the red blood cell content increased in all vessels simultaneously; 3) as the PIO2 decreased, the increase in red blood cell content occurred earlier in all vessels. Capillaries, therefore, can respond to hypoxia independently of the arteriole in cat cerebral cortical tissue.

Effect of hydroxynitrobenzylation of tryptophan-177 on reactivity of active site cysteine-25 in papain

Abstract It is known that the enzymatic activity of papain (EC 3.4.22.2) toward α-N-benzoyl- l -arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4-nitrophenyl (N-carbobenzoxy)glycinate ( S.-M. T. Chang and H. R. Horton, 1979 , Biochemistry18, 1559–1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactivation by SN2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloroacetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulfhydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the increased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, α-N-benzoyl- l -phenylalanyl- l -valyl- l -arginine p-nitroanilide, and toward an ester substrate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxynitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward α-N-benzoyl- l -arginine p-nitroanilide, apparently by blocking nonproductive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159).

The Intracellular Heterogeneity of Oxygen Concentrations as Measured by Ultraviolet Television Microscopy of PBA Fluorescence Quenching by Oxygen

If oxygen moves through the cell along special channels (Longmuir, 1977) and the mean solubility coefficient for oxygen in liver cells is rather low as Zander (1976) has shown, then there should be considerable intracellular heterogeneity of oxygen concentration even in a non-respiring cell with uniform PO2 throughout. Since the Polarographie electrode measures PO2 (Longmuir and Allen, 1960), this heterogeneity cannot be detected by this method. However, the fluorescence of pyrenebutyric acid (PBA) is quenched in proportion to the concentration of oxygen in a volume of a few hundred Augstrom’s radius around each photofluor molecule (Longmuir and Knopp, 1976). Thus, this method, with a theoretical geometric resolution of 0.2 μm appears to have this capability.

A New Histochemical Stain for Intracellular Oxygen

The observation of Vaughan and Weberl that oxygen concentrations in the physiological range would quench the fluorescence of pyrenebutyric acid in solution suggested to us that this principle might be used to measure intracellular PO2. Such a technique could eliminate the disadvantages encountered with techniques using electrodes, namely tissue damage and alteration of oxygen tensions by consumption of oxygen, providing that the following criteria could be fulfilled: (a) Penetration of the fluorescent probe into the cell. (b) Absence of metabolic damage as determined by respiration rate. (c) The quenching of the fluorescence of the intracellular probe by oxygen should occur according to a known relationship, preferably Stern-Volmer2.

THE QUANTITATIVE INTEPRETATION OF A/D PARAMETERS FOR ELECTRONIC EXCITATION MO ASSIGNMENTS OF TRANS-[M(CO)4(P(n-Bu)3)2] (M = Cr, Mo, AND W) BASED ON ELECTRONIC MCD AND COMPLETE OPERATOR MATRIX 〈Lz〉 EVALUATION

Abstract Electronic absorption (EA) and magnetic circular dichroism (MCD) spectra of trans-[M(CO)4(P(n-Bu)3)2] (M = Cr, Mo, W) of five bands from ∼20.0 × 103 to ∼45.5 × 103 cm−1 were measured. The EA spectra exhibit three dominant bands, I-III, at about 27 × 103 cm−1, 30 × 103 cm−1, and 39 × 103 cm−1, respectively. Corresponding MCD spectra in the region of both hands I and III are dominated by the presence of positive A-terms, indicative of transitions to degenerate excited states. A-terms and dipole strength (D) parameters were extracted by rigorous spectral deconvolution for the Cr complex; the spectra of the Mo and W analogs are quite similar. Since these are charge transfer bands and a number of the 2-center and 3-center integrals of 〈Lz〉 will be large, multi-zeta all-center operator matrix computations were used to evaluate signs and magnitudes of MCD A/D parameters for all degenerate excited-state electronic configurations of statically electric dipole allowed g → u (MLCT, or ·M → π∗(CO)) type tran...

A combination spectrophotometer for measuring electronic absorption, natural circular dichroism, and magnetic circular dichroism spectra

The design, construction, and evaluation of a combination spectrometer for measuring electronic absorption (EA), natural circular dichroism (CD), and magnetic circular dichroism (MCD) are described. Around the optical components of a JASCO ORD/UV‐5 spectropolarimeter, a new EA/CD/MCD instrument was built with the realized intentions of increasing sensitivity and upgrading the analog tube type circuitry to a solid‐state digitally, computer‐controlled spectrophotometer. It is a flexible, dynamic, and user‐controllable system, interfaced to an Apple II Plus computer, for studying instrument and signal parameters. The monochromator (M), photoelastic modulator (PEM), photomultiplier tube applied voltage (PMHV), and photomultiplier tube dc output current (PMdc) are under complete and independent software control. Our system has two unique aspects for obtaining the circular dichroism. First, the ac signal is measured with a voltage‐to‐frequency (V/f) converter; and, second, both the ac and the dc are independent...