mpt., 1 Y W
TRYPSIN MONOLAYERS AT WATER-AIRINTERFACES
1193
TRYPSIN MONOLAYERS AT THE WATER-AIR INTERFACE. I. FILM CHARACTERISTICS ANI) THE RECOVERY OF ENZYMATIC ACTIVITY BY B. ROGERRAYAND LEROYG. AUGENSTINE Contribution jrom the Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Ill. Received February 84# 1956
Trypsin films spread over a limited surface area of ammonium sulfate were studied as to film pressure-area and film preasure-concentration characteristics and the amount of recoverable enzymatic activity. .4Wilhelmy type balance employing electromagnetic control was used. A method for recovering films was developed whirh permitted a sensitive enzymatic: assay. It was found that film pressure and recoverable activity were very dependent upon the past history (maiiipulittionu) and the age of the film. No activity could be recovered from films which had previously existed a t surface concentrations below 6 ?/lo0 cm.2 I n more concentrated films, spread for 5 min., the recoverable activity corresponded to tho amount of enzyme spread in excess of 10 ?/lo0 cm.2 The recoverable activity decreased, independently of surface concentration, with increasing film age. The data suggest that the unfolded trypsin molecule has lost all activity and that a compressed film is composed of definite fractions of enzymc in globular and unfolded configurations.
Introduction I n recent years considerable interest has developed in the physico-chemical properties of proteins adsorbed at interfaces, principally the airsolid and air-water interfaces. An excellent review is that of Another direction of study has been into the effects of adsorption upon biological activity. Most of this work has been concerned with either proteolytic or antigen-antibody reactions carried out a t the interface. Rothen3 has summarized the research in this field. A number of reports have been made on the recovery of protein from a film followed by a determination of the effect that spreading had on the biological activity-in particular, enzymatic activity. Tho lack of experimental details in most of the published work is to be deplored. Gorter4 reported 8OY0recovery of activity from pepsin and trypsin films. However, no other quantitative data were given, so that the state of the films is unknown. Rothen, who was successful in recovering active insulin but not metakentrin and gonadotropic hormone, suggests that it is probably impossible to recover activity from a completely unfolded molecule. Langmuir and Schaefer6 recovered 80% of the activity of pepsin films after deposition on a slide. Several investigators6 have compressed mixed films of pepsin and albumin into threads and have measured the proteolytic activity of the threads. The recent work of Cheesman and Schuller7 is noteworthy for the care taken and, in addition, for. the details that are furnished. Using pepsin they recovered up to soy0activity. The evidence is, then, that activity can be recovered frorn monolayers of several daerent enzymes. But there is little known as to the critical conditions, Le., factors such as the manner and extent of spreading, age of film and method of recovery. The first objective of the present work (1) I o part from a thesis hy Leroy 0. Augenstine srihrnittrd to the Graduate Collage of the University of Illinois i n liartiill f~ilfillniento f the requirements for the Ph.D. Degree in Physico-Cheniical Biology, 1935. Additional experimental detail. can be f o u n d in the thesis. ( 2 ) H . Bull. Advance8 in Prolein Chem.. S, 95 (19.47). (3) A . Rothen. i b i d . , 3, 122 (1947). (4) E. Corter, Proc. R o y . Soe. ( L o n d o n ) . 166, 707 (1936). ( 5 ) I. Langmuir and V . Schaefrr. J . S m . Chein. Soc., 60, 1351 (1938). (6) (a) D . Ma:aia and G . Blumenthal. J . Cell. Comp. Physiol., 36, Suppl. 1 , 171 (19.50): ( b ) T. Haynshi and G. Ediaon. J . C o l l . Sei., 6, 437 (1950): (c) J. Kaplan, i b i d . . 7 , 382 (1952). (7) D . Checstnrrn and H. Schuller, i b i d . , 9 , 113 ( l < l 5 4 ) .
was to study these factors as they apply to trypsin. The behavior of proteins a t interfaces is obviously part of the general problem of elucidation of protein structure. There has been proposed already a “weak-link” hypothesis relating to protein reactivity‘vs; this has been utilized to interpret certain radiation effects upon proteinss in bulk. It is our feeling that additional information of value may be gained from investigation of the effects of radiation upon adsorbed protein. In particular, the effects of radiation upon film pressure and upon recoverable enzymatic activity seemed worthy of study. To this end, X-ray and ultraviolet irradiations of trypsin monolayers have been carried out. The present contribution forms the first of a series of three. Here we describe the physical charact,eristics of compressed monolayers of trypsin and the recovery of enzymatic activity. The second contribution presents the results of the irradiation studies; the last paper discusses the results and attempts an int,erpretation in terms of the “weak-link” concept.
Film Pressure Characteristics A . Experimental Methods Measurement of Film Pressure.-The apparatus was a modification of a Wilhemy-type balance described by Bull.* The novel features were the electromagnetic balanciiig arid the phot>oelectricdetermination of the end-point.’ Characteristics of this film balance are that readings are readily reproducible to 0.01 dyne/cm., it is capable of operation from a dist,anre (useful in radiation experiments), the determination of the “end-point” is reduced t o the simple ohservation of the deflection of a microammeter, and the complete operat,ion can be made automatic. Film troughs and harriers were made of Lucite and were heavily paraffined before use. The experiments were carried out inside a glass case which retarded evaporation and reduced contamination and thermal changes. I n esperinients extending over long periods of time the film trough was enclosed by close-fitting Lucite covers to eliminate evaporatioii. The work was done in an isolated basement room relatively free of organic vapors and dust, and constant as to temperitture over intervals of a day or so. Teniperature variation was less than +0.5’ for any one experimeiit, and all experiments were tietween 22 and 24O except where noted. 2 . Preparation of Solutions.-The support Polution was 15% ammonium sulfate to which 10 ml. of saturated sodium barbiturate (Veronal) had been added per liter of 1.
(8) L. Augenstine, “Information Theory in BioIogy,”ed. H. Quastler, Univ. of Illinois Press, Urbana, Ill., 1953, pp. 119-122. (9) E. Pollard, “Biochemical Aspects of Basic Mechanisms in Radiobiology.” National Research Council. No. 367. 1954 p. 1-29.
B. ROGERRAYA N D LEROYG. AUUENSTINE
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solution to control the pH a t 7.6. Merck Analytical Grade chemicals and conductivity water from a tin-lined still were used. After considerable investigation, the following standard purification procedure was adopted to remove surface-active contaminants from the support solution. After standing 6 hours, activated charcoal (Darco G-60) was added in the amount of 10 g./l. After 15 minutes the solution was twice vacuum filtered through medium and extremely fine filter papers. This charcoal treatment was repeated after 30 hours. The solution was then allowed to stand for at least 12 hours and was again vacuum filtered through fine filter paper just before being placed in the film trough. Well-washed, acid treated, analytical grade filter pa ers were employed. Frior to the spreading of enzyme, the support solution was swept t\vo or more times. (The only exceptions are the data in Fig. 1.) First, excess solution was swept off with a 1
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Fig. 1.-The effect of surface contnmination on film pressure-area plnt,s of ti,ypsin. The dashed curves, A, B, C, show the compression charaaterist,ics of three support solutions without rnzynie. The solid cut'ves, A4',B', C', refer t o the same solutions with 107 of trypsin spread on each. The initial area was 400 cni.2 in all cases. barrier; then small amounts of support solution were added and the surfnce reswept. This filtering and sweeping procedure gave a support solution which showed, on the average, a 0.25 dyne/cm. decrease in surface tension upon a 5 : 1 compression. A 5 : 1 cornpression means that a barrier was slowly moved from the end of the trough opposite the Wilheliny plate toward the plate to decrease the area fivefold. Our criterion of not more than 0.25 dyne/cm. surface pressure upon a 5 : 1 compression is less exacting than that of 0.15 dyne/cm. upon 8 : l compression recommended by Bull2 for force-area determinations. However, the present experiments were concerned wit,h measurements of film pressure of relatively compressed films rather than with force-area relations initiated at very large areas. The trypsin solutions were prepared in Pyrex which had been surface treated with a silicone (Desicote). Twice recrystallized, salt-free trypsin (Worthington Biochemical ill HCI. Co.) was sprinkled on the surf:ice of 1 X Without stirring, the solution R:LS kept in the refrigerator for 12 hours. The PI€ ~ n then s adjusted to 7.6 with Verona1 and the volume made u p to give a O.Ol?& solution of enzyme. Ten-nil. portioiis of this solut,ion were frozen in polyethylene vials and thawed as needed. The crystalline trypsin supply was stored at -7", and no loss in activity was observed during the course of the work. I n the pressurc-area determinations a fixed amount of protein solut,ion was onrefully placed dropwise on the surface using a Blodgett pipet delivering 0.0752 ml. In the pressure-concentration measurements variable amounts of protein solution were deposited from a 0.1-ml. graduated pipet. The enzyme concentrations were adjusted 80 that either one l3lodgett pipet or 0.1 ml. contained 10y of enzyme. Both pipets were Desicote-treated. B . Results 1. Effect of Surface Contamination on Pressure-Area Curves.-It is, of course, essential that the support solution be sufficiently free of contamination. Particularly, we were interested that a low degree of contamination be retained over the periods of time involved in the experiments.
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Repeated sweeping of the support solution was found to be inadequate by itself, perhaps partly due to slow diffusion effects. Extended treatment with activated charcoal plus sweeping was the most successful. Our experience with polyethylene bottles as containers for the solution has been that some were inert but that others introduced surfaceactive contaminants. The compression characteristics of surfaces possessing different degrees of containination are shown in Fig. 1 . Since the initial area in each case was 400 cm.2, the 5 : 1 Dashed curve A is typical compression point is a t 80 of the characteristics obtained with the standard procedure described above; dashed curves B and C resulted from inadequate charcoal treatment and either limited or no sweeping of the surface prior t o the measurements. The solid curves are the result of spreading trypsin on these three surfaces at an area and for periods of time corresponding to maximal spreading. As Bull2 has pointed out, an important check is whether pressure-area behavior is independent of the amount of protein. It was found that F-A plots for both trypsin and pepsin were independent over twofold increments in concentration whenever the initial spreading area exceeded 1.5 m.2/mg. and the time prior t o compression was of the order of 20 minutes. We define these conditions as being compatible with maximal spreading. The results were in excellent agreement with those reported by other investigat0rS.79'0 The solid curve A' in Fig. 1 is a portion of a representative F-A curve obtained with the regular procedures. 2 . Films Spread Over a Limited Area.-We found that films spread at large areas and then compressed had permanently lost all enzymatic activity. (The shortest length of time between spreading and compression studied was one minute). Therefore, our interest was directed t o films spread over limited rather than essentially infinite areas. The film pressure, measured 5 minutes after spreading a given aliquot of protein over the fived area of 100 cm.2, is plotted against surface concentration in each of the solid curves in Fig. 2. (The reciprocal function of surface concentration-film area-is given at the top .) Although film area is the traditional measure used by surface chemists, the surface concentration is the convenient measure in the present investigation. F I L M A R E A IN M ~ / M G IO 12 14
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Fig. 2.-The effect of various preparation procedures on film pressure-concentration plots of trypsin. Curves A, B and C refer to enzyme on support solutions which had been adequately treated with charcoal nnd filtercd 2 hours before use, 10 hours before use, and 48 hours hefoi,e use, respectively. Solutions were refiltered jnst prior to use. Curve D is a typical F-A curve obtained by spreading trypsin a t an area greaJer than 1.5 m.2/mg. for 20 min., or more, before compression. Such curves will be designated as F-C plots, as opposed to F-A plots. The difference lies in the conditions leading to the data: in F-A plots the amount of protein is held constant and the filni area is varied; in F-C plots the area is held constant and the amount of protein varied. Very different film Characteristics result. For example, the dashed curve D in Fig. 2 is an F-A plot, being the middle portion of curve A' from Fig. I. (IO) E. Mishuck and F. Eirich, "Monomolecular Layers," ed. Sobotka, A.A.A.S., 1954, pp. 14-32.
H.
TRYPSIN MONOLAYERS AT WATER-AIRINTERFACES
Sept., 1956
Pressure-concentration (F-C) plots were found to depend
to an important extent upon the temperature of the support solution, age of the film, and the elapsed time from the final charcoal treatment of the support solution. The effect of elapsed time from the final charcoal treatment is demonstrated in Fig. 2. The preparation procedure adopted was the one found to minimize the film pressure for a given surface concentration (curve C). Replicate F-C determinations had standard deviations as hi h as 10-15%. Since spreading involved the deposition of from 3 to 15 drops onto the surface and required 15-45 see., some of this variability was no doJbt due to the difficulty of reproducing the “dropwise” sequence. 3. Transitions between F-C and F-A Curves.-The film pressure a t any given area is, in many situations, dependent upon both the spreading conditions and subsequent manipulations (expansions or compressions) made prior to the measurement. To illustrate, the behavior of a typical film of trypsin carried through a series of compressions and expansions is summarized in Table I. Step 1 consisted of Hpreading 22y of enzyme upon an area of 40 cm.’. After two minutes the film was expanded (step 2) to 60 cm.2 and the film pressure measured 2 minutes later. The film was then expanded (step 3) to 80 cm.2 and allowed to age for 21 min. during which the pressure increased from 9.1 to 12.0 dyne/cm.
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(open circles) and those compressed to that area (CurveR). There is qualitative indication that expansion to a given area from a more limited area of initial spreading (dashed curves) is not equivalent to initial spreading a t the given area (solid curves).
TABLE I THEEFFECTS OF F ~ L M MANIPULATION A N D AGE O N THE FORCE-AREACHARACTERISTICS OF A TRYPSIN MONOLAYER Step no.
Type of manipulation
1 2 3
Spreading Expansion E:xpansion
4 5 6
Compression Expansion Expansion
7
Expansion
8
Expansion
9
Compression
10
Expansion
Age,of film since last preyious marupulation
2 2 3 5 10 15 21 2 2 2 4 8 2 4 7 2 4 1 2 6 2 4 3 2 2
IWm area, cm.2
Film pressure. dyne/cm.
40 60 80 80 80 80 80 70 80 100
>16.0 10.0 9.1 10.8 11.7 12.0 12.0 19.5 9.2 4.0 4.4 4.8 1.8 1.9 2.1 0.2 0.2 7.2 7.1 6.8
100 100 I20 1.20 120 160 160 100 100 100 160 160
0.1
0.1 6.7 300
