Anal. Chem. 1995, 67, 3336-3341
Direct in Situ Measurement of Phospholipid Hydration in an Aqueous Environment Using a Quartz Crystal Microbalance Kaori Wakamatsu,* Kasuo HosOda, Hiroshi Mitomo, and Masanao Ohya
Department of Biochemical Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376, Japan Yoshio Okahata and Kouichi Yasunaga
Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuda, Midori-ku, Yokohama, Kanagawa 227, Japan
Hydration of phospholipid head groups is essential for stabilizing bilayer structures in an aqueous environment. Here we describe a new, easy,and rapid method that can directly and quantitatively determine phospholipid hydration in both water vapor and liquid water; i.e., a quartz -tal microbalance is employedwhich enables hydration to be determined in only 1 h using just 1 pg of phospholipids under various environmental conditions. Dipalmitoylphosphatidylethanolamine(DPPE) was examined with varying temperature, KCl concentration, and phase of water, where it was found that DPPE hydration in liquid water markedly increases upon raising the temperature, even when the phospholipid is in a gel phase, being maximal around its phase transition temperature (63.5 "C). Furthermore, KCl addition increased DPPE hydration in a temperature-dependent manner. The method presented is demonstrated to be capable of determining phospholipid hydration under physiological conditions; hence, it is suitable for investigaling hydration phenomena involved in biological processes. Hydration of phospholipid head groups is essential for stabilizing bilayer structures in an aqueous environment. Since hydration
forces,'-> sometimes termed as hydration pressure, impede membrane close contact, hydration-dehydration processes are involved in the fusion and fission of biological membranes which occur during protein translocation withii cells or endocytosis. The amount of phospholipid hydration in water vapor can be directly determined via a classical gravimetric yet this technique cannot be applied to phospholipids suspended in liquid water. Instead, such hydration has only been quantified using somewhat timeconsuming methods, e.g., X-ray diffraction (XR.D),'jdifferential scanning calorimetry (DSC),' and 2H-NMR,s,gall of which use serial samples having increasing water/phospholipid ratios. (1) Rand, R. P. Annu. Rev. Biophys. Bioeng. 1981,10, 277-314. (2) McIntosh. T. J.; Simon, S. A Biochemisty 1986,25, 4058-4066 (3) Cevc, G.; Marsh, D. Biophys. J. 1985,47, 21-31. (4) Elworthy, P. H. J. Chem. Soc. 1961,5385-5389. (5) Jendrasiak, G. L.; Hasty, J. H. Biochim. Biophys. Acta 1974,337, 79-91. (6) Ruocco, M. J.; Shipley, G. G. Biochim. Biophys. Acta 1982,691, 309-320. (7) Chapman, D.Forms and Function of Phospholipids; Elsevier Scientific: Amsterdam, 1973; pp 117-142. (8) Finer, E. G.; Darke, A. Chem. Phys. Lipids 1974,12, 1-16. (9) Borle, F.; Seelig, J. Biochim. Biophys. Acta 1983,735, 131-136.
3336 Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
More recently, however, Okahata and Ebato'O showed that the adsorption of detergents onto a dimethyldioctadecylaoniuml poly(styrene-4sulfonate) membrane in an aqueous environment can be measured by using a quartz crystal microbalance (QCM).'O Their results showed that this adsorption was temperature dependent and reflected the membrane's phase transition. The QCM has also been successfully applied to examine the swelling behavior of Langmuir-Blodgett (LB) f i l m ~ , ~which l J ~ was found to be dependent on the film's phase.I3 These investigations led to the present study, which employs the QCM to quantitatively measure phospholipid hydration in liquid water. Our new method is unique in that only one phospholipid sample is needed to directly determine the resultant hydration within 1 h under given conditions, e.g., temperature and ionic strength. EXPERIMENTAL SECTION
Apparatus. Figure l a shows a schematic diagram of the QCM apparatus, comprising a probe made of an AT-cut (angle of cut which minimizes resonance frequency temperature depend e n ~ e quartz ~ ~ ) crystal disk (0.568 cm2;Kyushu Dentsu, Nagasaki, Japan) having a 0.2@cm2Au electrode deposited on each side, a laboratory-built power supply, and a frequency counter (SC 7201; Iwatsu) connected to a personal computer. The probe was siliconized using dimethyldichlorosilane in toluene and blocked with methanol to avoid possible hydration of the quartz crystal or gold electrodes, though nontreated probes gave essentially the same results. In measurements taken in a KC1 solution, to avoid current leakage between the two electrodes on the crystal via the ionic solution, one side of the crystal disk was covered with a plastic film. Without this shield of passivating film, the observed frequency fluctuated by > 1000 Hz. The power supply applied ac voltage across the electrodes to drive the quartz at its resonance frequency (9 MHz),I3 with the vibration frequency being subsequently measured by the frequency counter. The mass change (10) Okahata. Y.; Ebato, H. Anal. Chem. 1991,63, 203-207. (11) Okahata, Y.; Ariga, K. Thin Solid Films 1989,178, 465-471. (12) Ariga, IC; Okahata, Y. Langmuir 1994,10, 2272-2276. (13) Okahata, Y.; Kimura, K.; Ariga. K. J. Am. Chem. SOC. 1989,111, 91909194.
0003-2700/95/0367-3336$9.00/0 0 1995 American Chemical Society
ETl
Frequency counter
-
circuit
=&Jn I
1-(
computer
Quartz-crystal probe
I.To Thermostat
Oscillating
Thermometer
/d;ark-crystal
I
Sulfuric 1 acid
Distilled water
Au electrode
Figure I. Schematic of experimental QCM apparatus showing the
overall system and probe setup for vapor and liquid phase measurements. Am (g) can be calculated from the observed frequency shift AF (Hz) of an AT-cut, shear-mode QCM using14J5
Am =
-A
b+4q)0.5
2Ft
AF= -1.07
10-~ AF
(1)
where FOis the parent frequency of the quartz crystal (9 MHz), A the electrode area (0.20 cm2), eq the density of quartz (2.65 g ~ m - ~and ) , pq its shear modulus (2.95 x 10" dyn cm-9. In agreement with eq 1, QCM calibrations using palmitic acid indicated that a decrease in frequency (hF) of 1 Hz corresponds to an increase in electrode mass (Am) of 1.05 f 0.01 ng. This ratio (Am/hF) was subsequently employed to calculate Am. In preliminary experiments, the employed QCM and one made of a highly polished quartz crystal gave the same results, thereby eliminating the possibility that surface roughness contributed to the measured Materials. Dipalmitoylphosphatidylethanolamine (DPPE Avanti Polar Lipids Inc., Alabaster, AL) was used without further purification because it gives a single spot on silicic acid thin-layer chromatography. Water (HzO) was purified by a Milli-Q system (Millipore, Tokyo, Japan). We also employed DzO (99.9 atom % D; Isotec, Miamisburg, OH) in some control experiments, distilled prior to use, to confirm that the observed frequency change was solely due to the change in mass of the phospholipid film. Other reagents were of analytical grade (Wako, Osaka, Japan). Hydration in Water Vapor. To deposit phospholipids on the Au electrode, a chloroform solution ( 50 mM> reflects an increased space between the bilayers of the tilted DPPE molecules due to increased hydration. It is also important to realize that the extent of hydration enhancement due to KCl varies with temperature; i.e., the addition of 0.15 M KC1 (physiological ionic strength) increased DPPE hydration by a factor of about 5 at 20 "C, but only a factor of 2 at 40 "C. To the best of our knowledge, salts have not been reported to affect phospholipid hydration. Because similar QCM measurements showed no change in DPPC hydration upon the addition of KCl up to 150 mM (data not shown), the dependence of hydration on ionic strength may be specific to P E thus, the phenomenon has never been observed before. DISCUSSION
Validation of Measurements. In addition to the mass of adsorbate on the quartz probe, other factors will also affect the QCM's resonance frequency, e.g., sample viscoelasticity, surface roughness of the quartz, and interfacial slippage.16 However, we nevertheless consider these phospholipid hydration measurements to be accurate based on the following. (1) The resultant amount of adsorbed water was proportional to that of the phospholipid cast on the Au electrode (Figures 2b and 3b), a behavior that must occur for the frequency change to reflect solely the mass change.16 Such behavior also excludes the possible occurrence of slippage between phospholipid bilayers. In fact, under experimental conditions similar to those employed here, Okahata and Ebatoz5 showed that no slippage occurs between bilayers of 1,3dihexadecyl-2-glycerophosphoethanola mine, a phospholipid having a similar chemical structure. (31) Seddon, J. M.; Harlos, K.; Marsh, D. J. Biol. Chem. 1983,258,3850-3854.
3340 Analyfical Chemistry, Vol. 67, No. 78, September 75, 7995
(2) An impedance analysis of DPPE hydration in DW indicated that the resonance resistance (R) did not significantly change over the entire hydration time course (615.1 k 6.2 &2at 30 "C and 620.5 & 7.9 Q at 60 "C); thereby ruling out any possibility that viscoelastic changes in DPPE affect AF. (3) It is possible to contirm that the observed frequency change is due solely to the adsorption of water molecules into a phospholipid film by comparing the frequency change in H2O to that in D2O and obtaining a frequency ratio of 1.1 (molecular weight ratio of D2O and H20).32 In fact, when DzO was used instead of H20, AF due to hydration increased by a factor of 1.10 k 0.03 (n = 3) for DW and 1.108 i0.003 (n = 3) for 0.15 M KCl, indicating that A F reflects only a change in probe mass and not a viscoelastic change in DPPE16 (the mass of KCl does not contribute significantly to the mass ratio at 0.15 M). (4) The amount of hydration determined in this study is consistent with those determined using other methods (Table 1); e.g., the addition of cholesterol was found to increase hydration. (5) Measurement reproducibility is good since the SEM of hydrated water is as small as 1 mol/mol (n = 5) and 2 mol/mol (n = 3) for hydration in water vapor and liquid water, respectively. Advantages of Using the QCM for Studying Phospholipid Hydration. Hydration of phospholipids in liquid water has been determined by XRD,6,27.28 DSC? and 2H-NMR8jgmethods, all of which require multiple measurements using serial samples with increasing water/phospholipid ratios. The amount of hydration is obtained by determining the ratio where the interbilayer distance becomes constant in XRD, where the endothermic peak appears due to thawing of (free) ice in DSC, and where a sharp resonance of free water occurs in 2H-NMR In addition to the timeconsuming labor required for these techniques, DSC has another drawback in that it can determine the hydration only at 0 "C. The presented QCM method, however, possesses many clearcut advantages. (1) It is straightforward and monitors a frequency change that is directly proportional to the phospholipid mass change. (2) Hydration is rapidly determined (< 1h) using just a single sample under given conditions such as ionic strength and temperature. (3) Only 1pg of phospholipids is required, which makes it possible to analyze pathological samples that may be available in only limited amounts. (4)Temperature of a sample can be easily controlled. (5) Monitoring the time course of hydration is possible at a resolution of 1s.12 (6) The QCM method can be applied in both gaseous and liquid environments that are close to physiological conditions. CONCLUSIONS The advantages of using the QCM to quantitatively measure phospholipid hydration enabled us to systematically study such hydration in liquid water under various environmental conditions, as well as to directly compare hydration in water vapor with that occumng in liquid water. Therefore, the QCM is expected to become a promising tool for studying the hydration of biological molecules under physiological conditions. In fact, our new method is believed to be generally applicable to research on the (32) Lasky,S. J.; Buttry, D. A. j . Am. Chem. SOC.1988, 110, 6258-6260. (33) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21-51. (34) Mason, W. P. In Physical Acoustics: PrinciPles and Methods, Volume I, Part A; Mason, W. P., Ed.; Academic Press: New York, 1964; Chapter 5.
solvation of any other compound that can be fixed onto an Au electrode.
are grateful to Dr. Toshiaki Dobashi, Gunma University, for his valuable technical advice.
ACKNOWLEDGMENT
Received for review January 26, 1995. Accepted June 21,
This work was financially supported by a Grant-in-Aid (No. 03780145) from the Ministry of Education, Science, and Culture of Japan, and by a grant from the Asahi Glass Foundation. We
AC950098D
1995.B
@
Abstract published in Advance ACS Abstracts, August 1, 1995.
Analytical Chemistry, Vol. 67, No. 18, September 15, 1995
3341
