Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

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Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene
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  Oxygen Diffusion in Biological and Artificial Membranes Determined by the Fluorochrome Pyrene STEVEN FISCHKOFF and J.M. VANDERKOOI From the Johnson Research Foundation, Department of Biophysics and Physical Biochemis- try, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19174 ABSTRACT Quenching of pyrene fluorescence by oxygen was used to deter- mine oxygen diffusion coeffÉcients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80 % in the presence of 1 atm O,, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37°C are 1.51 X 10 5 cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10 6 cm~/s in dipalmitoyl lecithin vesicles, and 7.27 X I0 -s em2/s in erythroeyte plasma membranes. The heats of activation for oxygen diffusion are low (< 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed. Knowledge of oxygen diffusion rates in tissue is essential in evaluation of the role of oxygen in control of metabolism. Oxygen diffusion coefficient in skele- tal muscle was found to be 1.3 X 10 -5 cm2/s by Krogh (1929). This value closely resembles the oxygen diffusion coefficient in other tissues, and in blood plasma (Hartridge and Roughton, 1927) and erythrocytes suspension (Forster et al., 1957; Klug et al., 1956; Thews, 1968). The similarity in the oxygen diffusion coefficient between tissue and protein solutions is generally taken to mean that the membrane imposes no barrier for oxygen (Boag, 1969; Wittenberg, 1970). In this paper we attempt critical analysis of this assumption. Quantitative determination of the oxygen diffusion constant is afforded by use of the diffusion-limited reaction between the para- magnetic species oxygen and the singlet excited state of a fluorescent probe molecule located in the membrane. Pyrene is chosen as probe because its in- THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 65, x975 • pages 663-676 663  onD  e c  em b  er 1  ,2  0 1  5  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published May 1, 1975  664 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 6 5 . i975 trinsic fluorescent lifetime is long, thus collisions between excited state pyrene and oxygen cause significant quenching of fluorescence. Furthermore, pyrene is a lipophilic hydrocarbon which readily partitions into the lipid phase of the membrane, and molecular pyrene diffusion has been previously described (Vanderkooi and Callis, 1974). Derivatives of pyrene have been successfully used to determine the presence of oxygen in proteins (Vaughan and Weber, 1970) and in rat liver cells (Knopp and Longmuir, 1972). MATERIALS AND METHODS Chemicals Pyrene (Eastman Chemical Company, Rochester, N. Y.) was twice recrystaUized from ethanol and was determined to be free of fluorescence impurities by a single exponential fluorescence decay over three log units in ethanol. Sigma Chemical Company (St. Louis, Mo.) supplied DL-a-dipalmitoyl lecithin (lot no. 93C-2900) and cholesterol (lot no. 106B-1630). L-a-dimyristoyl lecithin (lot no. 9434) was supplied by Nutritional Biochemical Corporation (Cleveland, Ohio). Prepurified oxygen and nitrogen were obtained from Air Reduction Company (New York). All other reagents were of the highest purity commercially available. Twice-glass- distilled water was used throughout. Preparation of Membranes Artificial phospholipid membranes were prepared by sonicating the lecithin and pyrene in phosphate-buffered aqueous solution (pH 7.4) at around 45°C for 3-5 rain on a Branson sonifier (Branson Sonic Power, Danbury, Conn.). Erythrocyte plasma membranes from human blood were prepared by the procedure of Dodge et al. (1963), and the protein concentration was determined by the biuret method (Gornall et al., 1949). Pyrene was incorporated by adding a concentrated alcoholic pyrene solution to aqueous suspension of membrane. The fluorsecence intensity of the pyrene-membrane samples was stable 30 s after pyrene addition, indicating that the partitioning of pyrene into the membrane phase occurred within this time. Fluorescence Measurements Steady-state fluorescence was measured with an Hitachi MPF-2A fluorescence spectrometer (Hitachi Ltd., Tokyo, Japan) using conventional 90 ° optics. The sample was maintained at the desired temperature with the use of circulating water through the cell block. Relative quantum yields of the samples were determined using 386 nm as emission wavelength and 335 nm as excitation wavelength. The decay of fluorescence was measured on an Ortec photon-counting fluorescent lifetime instrument (EGG. Ortec, Oak Ridge, Tenn.), as previously described (Van- derkooi and CaUis, 1974). Temperature control was achieved using a thermoelectric module (Temptronix Company, Needham, Mass.)with thermostatic control incorp- orated into the sample cell block. Data were analyzed for exponential decay by  onD  e c  em b  er 1  ,2  0 1  5  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published May 1, 1975  FISCrmOFF AND VANDERXOOI Oxygen Diffusion in Membranes 665 nonlinear regression on a PDP-10 computer, also as described previously (Van- erdkooi et al., 1974). The fluorescence measurements were made using a cuvette with a screw-type cap equipped with a rubber septum (Hamilton Company, Reno, Nev. type 18288) through which a syringe could be inserted to allow addition of gas. Nitrogen and oxygen in the desired ratios were mixed before bubbling by use of a Matheson gas mixer, model no. 7322 (Matheson Co., Inc., East Rutherford, N.J.). The suspensions (3-4 ml) were equilibrated with each gas mixture by bubbling the gas through the suspension for 15 rain at a rapid rate, followed by removal of the syringe. Longer times of bubbling did not change the fluorescence lifetime or yield, indicating that equilibration had occurred. The fluorescent lifetimes were constant over a period of 6 hr, which was taken to mean that leakage of the gases to the atmosphere did not occur. The oxygen concentration in the medium was calculated by use of litera- ture values of oxygen solubility in aqueous solutions (International Critical Tables). Since the rate of reaction between excited-state pyrene and oxygen is first order with respect to oxygen concentration (Eq. 4) it is necessary to know the oxygen con- centration in the membrane in order to calculate the diffusion constant. In our experiments this value is unknown, and we have calculated the diffusion coefficient assuming oxygen to be in the same concentration in the membrane as in the aqueous medium. A more reasonable estimate of oxygen concentration is to use the distribution coefficient for membrane/water of 4.4, determined by Battino et al. (1968), and using this value, the diffusion coefficient was also calculated. In all experiments, pyrene concentration was kept low so that pyrene excimers were not formed (Vanderkooi and Callis, 1974). The absence of excimers was con- firmed by the absence of fluorescence emission at 470 nm. This precaution is neces- sary since excimer formation competes with oxygen for the quenching of excited- state pyrene monomer, thus complicating analysis. CALCULATION OF DATA The relevant processes involved in determination of diffusion constants from fluores- cence quenching data are given below: P + hu --~ P* (absorption of light), p, kj ~ P + hv (fluorescence emission), (1) (2) P* [- 02 k , (quenching), (3) where P and P* represent the respective ground and excited states of the fluorescent molecule, i.e., pyrene, and k I is the rate of fluorescence decay, which is related to fluorescence lifetime, r by 1 k -- -. (4) T  onD  e c  em b  er 1  ,2  0 1  5  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published May 1, 1975  666 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 6 5 • ~975 The biomolecular rate constant, k, for O, quenching of pyrene fluorescence can be obtained from the Stern-Volmer Equation (Stern and Volmer, 1919): F0 T0 F T - 1 + kr0[o2], 5 ) where Fo and ro are the fluorescent intensities and lifetimes in the absence of quencher and F and z are the intensities and lifetimes in the presence of quencher. Making the assumption used by others (Osborn and Porter, 1965; Lakowicz and Weber, 1973) that every collision between P* and oxygen is effective in quenching, the rate con- stant, k, is then related to the diffusion constant, D, by the Einstein-Smoluchowski expression: k = 47rRDN', ( 6 ) where N' is Avogadro's number per millimole, R is the sum of the molecular radii, and D is formally the sum of the diffusion coefficients for oxygen and pyrene. In our calculations we assumed that D is the diffusion coefficient of oxygen alone, a reason- able assumption on the basis that the diffusion coefficient of oxygen is much larger than for pyrene. We further assumed that the interaction radius, R, is 6 X 10 s cm, an assumption which is based on the molecular sizes of pyrene and oxygen. Since the fluorescence yields and lifetimes are related to the rate, k, (Eq. 4) the heat of activation (E,) for oxygen diffusion can be calculated by two methods, Arrhenius plots of fluorescence intensity (F) or fluorescence lifetime (~-) versus temperature: -- 2.303R T + Ca, ( 7 ) or log ) - 2.303R-------W + C2, 8 ) where T is the absolute temperature, R is the gas constant, and Ca and C~. are con- stants. Both these methods were used to determine E~ as a check for the internal consistency of the measurements. RESULTS Fluorescence Emission Parameters of Pyrene The fluorescence yield of pyrene incorporated into phospholipid vesicles is quenched by either complex formation between ground-state pyrene and oxy- gen, (Evans, 1953) or alternatively, between excited-state pyrene and oxygen as described in Eqs. 1-3. That the second mechanism accounts for the fluores- cence quenching is supported by several lines of evidence: (a) The absorption spectrum of pyrene in ethanol is unaltered by the presence of atmospheric  onD  e c  em b  er 1  ,2  0 1  5  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published May 1, 1975  FISCHKOFF AND VANDERKOOI Oxygen Diffusion in Membranes 667 03. (b) The fluorescent lifetime, r, is decreased by the presence of oxygen and the decay is exponential, as seen by the linear relationship between the loga- rithm of fluorescence intensity and time (Fig. 1). (c) The fluorescent yield of pyrene but not its excitation and emission spectra is affected by the presence of oxygen in the sample. These phenomena are in agreement with the reaction mechanism given in Eqs. 1-3 and with the Einstein-Smoluchowski equation for diffusion given in Eq. 6. Diffusion Coefficient or Oxygen in Membranes The fluorescent lifetimes for pyrene were measured in different phospholipid vesicles or membranes as a function of O, concentration and temperature (Table I). The lifetime of pyrene in the absence of the quencher, oxygen, was around 200 nl at 25°C for all membranes, and as the temperature was raised, the lifetime decreased. These data are plotted according to the Stern-Volmer relationship (Eq. 4) in Fig. 2. The slope of these plots yields the rate constant for the reaction of oxygen with pyrene, and allows calculation of diffusion coefficients (Eq. 5). A summary of the results is presented by Table II. The diffusion constant is pre- sented assuming oxygen concentration in the membrane to be the same as in the aqueous medium (third column) or more realistically to be more concen- trated in the membrane, (last column). The diffusion coefficient of oxygen in phospholipids agrees closely with that in red blood cell membranes, and is close to literature values in water or tissue. This finding suggests that the diffusion 4 3 ~.~ o~ ~ I00 ns ~ FmuRE 1. Fluorescence decay of p~ene in dimFristoyi lecithin ve~cI~. The samples contained 0.3 nag dimyfistoy lecithin/m1, 0.1 #M py~ne, and 10 mM PO4 buffer, pH 7.4. The samples were equilibrated with gases indicated on the figure, and fluores- cence decay curves were obtained as described in Materials and Methods. Temperature was 25°C.  onD  e c  em b  er 1  ,2  0 1  5  j   g p.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published May 1, 1975
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