Langmuir 1993,9, 574-576
674
In Situ Surface-Detecting Technique by Using a Quartz-Crystal Microbalance. Interaction Behaviors of Proteins onto a Phospholipid Monolayer at the Air-Water Interface' Yasuhito Ebara and Yoshio Okahata' Department of Biomolecular Engineering, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152, Japan Received July 10, 1992. In Final Form: November 16,1992
Interaction of various proteins adsorbed from solution with a phospholipid monolayer at the air-water interface of a Langmuirfilm balancehas been studied quantitativelyby wing a quartz-crystal microbalance (QCM) which was attached horizontally on the monolayer from the air phase. Adsorption amounts and penetration behaviors of proteins have been followed by observing frequency changes of the QCM at the air-water interface and surface pressure changes of the monolayer. Introduction Interactions of proteins with cell membranes are of wide interest in studies such as molecular recognition at cell surfaces. A monolayerlipid film at the air-water interface has been demonstrated to be useful in cell surface modeling. Studiesin adsorption behavior of proteins from solution with a lipid monolayer have been reported by using various in situ surface-detectingtechniques and dry processes:2 surface tension measurementa?~~ surface plasmon resonance? fluorescent-labeling spectra,- ellipsometry, and radiolabelingte~hniques.~*~ These methods have future potential in observingprotein adsorptions;however, it is still not clear how the interaction process of proteins with the interface can be understood.2 The former methodologiesrequire large and expensive equipment for in situ measurementa and have someaseociatsd difficulties to obtain quantitatively the amount of protein adsorbed and time-course of the adeorption process. In this paper, we report a new, easy,and in situhhnique to detect interactionsof proteins adsorbedfrom a subphase with a phospholipid monolayer of a Langmuir film balance, on which a quartz-crystal microbalance (QCM) was horizontally attached to the lipid monolayer from the air phase (see Figure 1). QCMs are known to provide very sensitive mass measuringdevices because their resonance frequency decreases upon the increase of a given mass on the QCM in the nanogram level. Adsorption and penetration behavior of proteins could be observed quantitatively from the frequency changes of the QCM on the monolayer (AZ9 and the surface pressure changes of the monolayer (Ar),responding to the addition of proteins. The amount of adsorbed proteins (Am)was also obtained from the frequency changes after lifting and drying in air. ~~
~
~~
(1) Characterizationof Langmuir-Blodgett Films. Part 14. For Part 13,eee: Okahata,Y.;Ebara,Y. J. Chem. SOC.,Chem. Commun. 1992,116. (2) Norde, W. Ado. Colloid Interface Sci. 1986, 25,261. (3) Watanabe, N.; Shirakawa, T.; Iwahashi, M.; Seimiya, T. Colloid Polym. Sci. 1988,266, 254. (4) Ivanova, M.; Panaiotov, I. Colloid Surf. 1986, 17, 159. (5) Kooyman, R. P. H.; Bruijn, H. E.; Eenink, R. G.; Greve, J. J.Mol. Struct. 1990,218, 345. (6) Blankenburg,R.;Meller, P.;Ringdorf, H.; Salesee, C. Biochemistry 1990,28,8214. (7) Heckl, W. H.;Thompeon, M.; Mtihwald, H.Langmuir 1989,6390. (8) Heyn, S.-P.; Egger, M.; Graub, H. E. J. phys. Chem. 1990,94,5073. (9) Brash, J. L.;Uniyal, S.;Pueineri, C.;Schmitt,A. J.Colloidlnterface Sci. l969,96, 28.
Protein t
Surface
pressure
JQ~M
It
I
c%(cbh,-%_.&+NW CMCYho-
0
2C, IPE
Figure 1. An experimentalsetupof a QCM attachedhorizontally on a 2C13Ephospholipidmonolayer of aLangmuir film balance. Experimental Section The QCM employed is commercially available 9-MHz, ATcut quartz (9 mm diameter)on both sides of which Au electrodes were deposited (16 mm2area). The QCM was connected to a handmade oscillatordesigned to drive the quartz at ita resonance frequency at the air-water interface.lh12 The frequencychanges were followed by a universal frequency counter (Iwatsu Co., Tokyo, Model SC 7201) attached to a microcomputer system (NECCo., Tokyo, Model PC 9801). Calibrationwas done by the deposition of 2-20 layere of lipid Langmldu-Blodgett (LB)f i b on the QCM,lhl*and the constant obtained was well consistent with the Sauerbrey equation in both air and water phases. Thus, a frequency dwreaae of 1 Hz corresponded to a mass increase of 1.05 ng on the QCM electrode (16 mm2).1h12 -AF = (1.05 f 0.01) Am (1) A monolayer of 1,3-dihexadecylglycero-2-phosphoetha1iolamine (2C13E)lwas spread on Milli-Q water (pH5.8)in a Tefloncoated trough with a microcomputer-controlled Teflon barrier (San-Esu Keisoku Co., Fukuoka).10J1J4 The stable monolayer formation was confirmed from surface preeeure (*)-area (A) isotherms at 20 O C . A QCM plate was attached horizontally on the 2ClePE monolayer at surface pressures of 10-40 mN m-l and the frequency changes of the QCM responding to the addition (10) Okahata, Y.; Ariga, K. Langmuir 1989,5, 1261. (11) Okahata, Y.; Kimura, K.; Ariga, K. J . Am. Chem. SOC.1989,111, 91Qn. (12) Okahata, Y.; Ebato, H. AM^. Chem. 1991,63,203. (13) Sauerbrey, G. Z. Phys. 1969,156,206. (14) Ariga, K.; Okahata,Y. J. Am. Chem. SOC.1989, 111,5618.
0 1993 American Chemical Society
Langmuir, Vol. 9, No.2, 1999 575
Surface-Detecting Technique
L 1 0 mN m.'
40 mN m-'
40 mN m.' k 10 mN m.'
0
10
20
I
30
Time I min Figure 2. Time-courses of surface pressure changes ( A d and frequency changes (AF)of the QCM on the 2ClePE monolayer at surface pressures of 10 and 40 mN m-l, responding to the addition of mellitin (100ppm, 35 pM, M, = 2840) from aqueous solution (pH 5.8, 20 "C).
Time I min Figure 3. Time-courses of surface pressure changes (AT) and frequency changes (AF) of the QCM on the 2Cl8E monolayer at the surface pressure of 10 and 40 mN m-l, responding to the addition of &globulin (100ppm, 1 pM, M, = 105) from aqueous solution (pH5.8, 20 "C).
of proteins from aqueous solution were followed with time. Surface pressure changes (AT) responding to the addition of proteins at the constant molecular area of the monolayer were also monitored.
rangeof lC300ppm (3.5-105pM1, theamountofadsorbed protein showed a saturation curve above the concentration of 100 ppm (35 pM). When the relativelylarge protein of &globulin (100ppm, 1pM, M, = 105)was injected into the solution, the similar AF and AT changes were observed as shown in Figure 3. However, the increase of AT at the low surface pressure of 10 mN m-l was smaller than that for the addition of mellitin (see Figure 2). When mellitin or fl-globulinwas added into the subphase solution (100 ppm, 35 pM or 1 pM, respectively), surface pressure changes of the monolayer (AT), frequency changes at the monolayer (A29 and the adsorbed amount (Am' ) calculated from AF,and the adsorbed amount (Am) calculated from the frequency changes after drying in air are summarized in Table I. In the case of relatively small and hydrophobicmellitin (Mw= 2850), the surfacepressure of the 2ClsPE monolayer increased largely responding to the addition of mellitin at the low surface pressure of 10 mN m-l but did not change at the high surface pressure of 40 mN m-l (see Figure 2). This indicates that the small and hydrophobic mellitin molecule adsorbs and penetrates into the lipid monolayer at the low surface pressure of the monolayer; on the contrary, it adsorbs near the surface at the high surface pressure of the monolayer. Adsorption amounta of mellitin calculated from frequency changes in both aqueous solution (Am' ) and dry in air (Am) were consistent with each other independent of surface pressure of the monolayer. These phenomena were observed for similar small and relatively hydrophobic proteins such as adrenocorticotropine (Mw 4500) and @-endorphin(M,3400). When the relatively large protein of @-globulin(M,= 105)was injected into the solution, the increase of AT at the low surface pressure of 10 mN m-l was smaller than that for the addition of the small protein of mellitin (see Figure 3). This indicates that the smaller and more hydrophobic mellitin can penetrate easily into the lipid membrane, but the larger &globulin cannot penetrate as easily. A schematic illustration is shown in Figure 4. In the case of the ,%globulininteraction, adsorption amounta of proteins obtained from frequency changes at the interface (Am' ) were 1.5times larger than those obtained in the dry state in air (Am), at surface pressures of both 10 and 40 mN m-1 (see Table I). The relatively large &globulin adsorbs near the surface of the lipid membrane and the QCM plate on the monolayer vibrates the adsorbed proteins with surroundedwater. Therefore,the frequency decrease was estimated in a larger value than those in the air phase or those when the small mellitin proteins
Results and Discussion When the QCM plate was attached horizontally on the water subphase, the frequency decreased 4350 f 10 Hz compared with in air, which was consistent with the theoretical frequency changes calculated from Kanazawa's equation considering viscosity and density of media (water).l5 The frequency difference between when the QCM attached on the 2ClsPE monolayer at 40 mN m-l and on the water subphase was observed to be -(50 f 5) Hz (the mass increase of 44 f 5 ng according to eq 1,which was well consistent with the mass of the monolayer under the electrode (16 mm2) of the QCM calculated from the molecular area of T-A isotherms. Figure 2 shows typical time courses of surface pressure changes (AT) of the 2ClsPE monolayer at the constant area and frequency changes ( A 0 of the QCM on the monolayer, responding to the addition of mellitin (100 ppm, 35 pM, Mw = 2850) from solution. The resonance frequency when the QCM was attached on the 2 C d E monolayer at the air-water interface was defined as standard (zero position). At the surface pressure of 40 mN m-l, the frequency of the QCM decreased (mass increased) gradually responding to the addition of mellitin and saturated at -AF = 105 f 10 Hz (Am = 100 f 10 ng according to eq 1) within 10 min. After reaching the equilibrium, the QCM plate with a lipid monolayer and adsorbed proteins were transferred to the air phase with a cover by a horizontal lifting method and then dried.16 We have confirmed that all lipid molecules and proteins adsorbed under the QCM could be transferred quantitatively as a monolayer in this method. The adsorbed amount of proteins was calculated to be Am = 100 f 10 ng by deducting the amount of lipid monolayer (44 f 5 ng), from the frequency change in the air phase before and after depositions. This value was consistent with the adsorption amount (Am' = 100 f 10 ng) calculated from "lues at the &water interface by eq 1. The amount of adsorbed mellitin obtained from the QCM method was consistent with the calculated amount of Langmuir-type adsorption of mellitin as a monolayer. When the concentration of mellitin in the subphase increased in the (15) Kanazawa, K. K.; Gordon, J. G . Anal. Chim. Acta 19M,1 7 6 9 9 . (16) Okahata, Y.;Ariga, K.; Tanaka, K. Thin Solid F i l m 1992,210/ 21 1,702.
576 Langmuir, VoZ. 9, No. 2, 1993
Ebara and Okahata
Table I. Surface Pressure Changes ( A T ) and Frequency Changes (AF) at the Monolayer-Water Interface Responding to the Addition of Proteins
proteinsa
initial surface pressure (mN m-*)
mellitin
10 40 10 40
8-globulin
AT
(mN m-l) 20 f 0.5 0 7.5 f 0.5 0
at the air-water interface AF Amtb (Hz) (ng) 105 f 15 100 f 15 80 f 15 76 f 15 256 f 15 244 f 15 290 f 15 276 f 15
after drying in air Ame (ng) 100 f 10 75 f 10 150 f 10 205 f 10
a Proteins were added 100 ppm in the subphase; [mellitinl = 35 p M and [8-globulin] = 1 p M . The adsorption behavior saturated in this concentration range of proteins. The amount of adsorbed protein calculated from the frequency change (AI?)at the air-water interface using eq 1. The amount of adsorbed protein calculated from the frequency change after drying in air using eq 1.
p-globulin
When proteins were injected into the aqueous phase at the high surfacepressure of 40 mNm-l, the surfacepressure was hardly changed independent of the size of protein molecules, although proteins were confirmed to adsorb on the surface of the monolayer from the frequency changes of the QCM. This indicates that it is dangerousto observe the protein interaction onto the monolayer only from the AT values, especially at the high surface pressure.
Figure 4. A schematic illustration of interactions of proteins with a phospholipid monolayer.
Conclusion
penetrated to the membrane. The Am value (150-200 ng) of @-globulinobtained after drying in air was consistent with the calculated amount of Langmuir-type adsorption of @-globulinas a monolayer,which seems to indicate the true amount of adsorbed @-globulin. When the concentration of @-globulinin the subphase increased in the range of 10-200ppm (0.1-2 pM),the amount of adsorbed protein showed a saturation curve above the concentration of 50 ppm (0.5 pM).Similarphenomenawere observed for other large proteins such as bovine serum albumin (M,= 7 X lo4)and concanavalin A (M, = 105).
The QCM system which attached horizontally to the lipid monolayer at the air-water interface will become a useful, new technique to detect quantitatively interaction behaviors of proteins with a lipid monolayer from the AF value at the interface, Am value after drying in air, and AT value of the monolayer. We are developingthis system to detect molecular-selectiverecognition phenomena between proteins and the lipid membrane interface such as the combination of the glycolipid monolayer with concanavalin A or the biotin-lipid monolayer and avidin proteins.
at 40 mN m-’
at 10 m N m”
Mellitin
