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Quantitation of a neutral‐buoyancy assay (NBA) to estimate transmembrane N 2 flux
Author(s) -
Moss Fraser J.,
Zeise Brian,
Huffman Dale,
O’Neill Steven,
Boron Walter F.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06055
Subject(s) - buoyancy , oocyte , bubble , chemistry , biophysics , neutral buoyancy , subcooling , analytical chemistry (journal) , chromatography , thermodynamics , mechanics , biology , physics , microbiology and biotechnology , boiling , embryo , organic chemistry
Several assays exist for CO 2 , O 2 and NH 3 permeability, but a major technical void has been the absence of an assay for the transmembrane movement of N 2 and other relatively inert gases. Here, we report the quantitation of a novel and simple yet powerful assay based on the neutral buoyancy of a Xenopus oocyte injected with a bubble of nitrogen (N 2 ) gas. Briefly, we inject a precise volume of N 2 (200 nl)—with a known number of gas molecules (n Gas )—creating a bubble that thereby lowers cell density (ρ Cell ) sufficiently for the oocyte to float. After injection, we transfer the oocyte to a glass tube (rated to 10 ATM), nearly filled with saline. We then pressurize the atmosphere above the saline solution sufficiently for the bubble inside the oocyte to constrict, thereby raising ρ Cell (decreasing buoyancy) and causing the oocyte to descend to a neutrally buoyant depth of 5 cm. As N 2 diffuses out of the bubble, dissolves in the surrounding cytoplasm, and eventually exits the oocyte, the tendency is for the bubble to collapse gradually (increasing ρ Cell ) and for the oocyte to sink. Rather than allowing the oocyte to sink substantially, we developed a neutral buoyancy clamp system (high‐resolution camera, software developed in‐house, digitally controlled pressure regulator) to adjust and record continuously the pressure in the air phase (P NB ) required to maintain neutral buoyancy at an oocyte depth of 5 cm. The pressure inside the bubble (P Bubble > P NB by a fixed amount) falls approximately exponentially over time, and proportionally with the decreasing n Gas . In order to calculate n Gas from the recorded P NB , we performed a series of experiments in which—for each oocyte—we measured the spontaneous initial oocyte volume before bubble injection (V i,Xo ), injected a known volume of N 2 (V Gas ), and then measured initial P NB for that combination of V i,Xo and V Gas . We repeated this procedure for 295 oocytes with V i,Xo ranging from 812 to 1590 nl, and V Gas ranging from 50 to 350 nl. For V Gas from 100 and 300 nl, we observed a linear dependence of P NB on V Gas /V i,Xo , with the best‐fit equation: P NB = 11.28*(V Gas /V i,Xo ) – 0.05 (Eq.1), R 2 = 0.95. Here, the units of P NB , the slope, and the intercept are ATA. Recalling the ideal gas law and inserting the expression for V Gas from Eq.1, we conclude that the apparent n Gas = P B *V i,Xo *(P NB + 0.05)/(11.28*RT) (Eq.2). Here, P B is room barometric pressure, and all units are consistent with an R of 0.0821 L atm K –1 mol –1 . Because of unavoidable experimental error in V i,Xo and V Gas , and variability in oocyte density, the apparent n Gas at time = 0 does not always match 8.26 nmol, the value computed for 200 nl of injected gas in our standard assays. We therefore rearranged Eq.2 to replace the best‐fit y‐intercept (i.e., 0.05 ATA) with an adjusted y‐intercept ( b ) that is characteristic for each oocyte. Using the revised relationship, n Gas = P B *V i,Xo *(P NB + b )/(11.28*RT) (Eq.3), we are now able to obtain the actual decay in n Gas during our standard NBA and estimate absolute transmembrane N 2 fluxes. While specifically developed for N 2 fluxes, the NBA should be applicable to measuring transmembrane fluxes of other gasses. Support or Funding Information Office of Naval Research (N00014‐15‐1‐2060 & N00014‐16‐1‐2535)