TRYPSIN INHIBITORS OF HUMANSERUM. I. STANDARDIZATION,MECHANISMOF REACTION, ANDNORMALVALUES'

By HALLIE F. BUNDY2 AND JOHNW. MEHL

(From the Department of Biochemistry and Nutrition, University of Southern California andthe Laboratories of the Los Angeles County General Hospital, Los Angeles, Calif.)

(Submitted for publication August 3, 1957; accepted March 6, 1958)

Camus and Gley (1) appear to have been thefirst to observe that human serum inhibits trypsin,and Ascoli and Bezzola (2) and Brieger and Treb-ing (3), the first to report that this trypsin in-hibiting activity may be increased in some diseases.Since that time the inhibition of trypsin by theserum and plasma of normal and diseased personshas been the subject of several investigations.Grob (4) and Jacobsson (5) have reviewed theearlier literature.

A variety of substrates and trypsin preparationshave been used in measuring the inhibition of tryp-sin by serum and in studying the mechanism ofthis reaction. The determination of inhibitor lev-els has received the most attention and resultshave often been expressed in arbitrary units orreferred to the amount of inhibition produced bynormal serum. Concentrations in normal humanserum have been expressed in terms of microgramsof crystalline trypsin inhibited per ml. of serumby Christensen (6), Peacock and Sheehy (7),Shulman (8), and Jacobsson (5). The averagevalues were 655, about 1,400, 2,200, and 752, re-spectively. In each case the assay method involvedpreincubation of trypsin and serum in an alkalinemedium. Christensen and Jacobsson, followingthe suggestion of Kunitz (9), standardized theirtrypsin preparations by titration with crystallinesoybean inhibitor.

Hussey and Northrop (10) and Grob (4),using crude trypsin preparations, found the inhi-bition by human plasma to be reversible. On theother hand, Shulman (11) and McCann and Las-kowski (12) using crystalline trypsin prepara-tions, the former with human serum and the latter

1 This investigation was supported by Research GrantRGA-269, from the National Institutes of Health, UnitedStates Public Health Service.

2 Taken, in part, from a thesis submitted in partial ful-fillment of the requirements for the degree of Master ofScience, Graduate School, University of Southern Cali-fornia.

with rat plasma, found the inhibition to be stoichi-ometric and irreversible. It had been recognizedthat there are two components in human plasmawhich inhibit trypsin, and Jacobsson (13) showedthat these could be demonstrated by zone electro-phoresis to be components of the a,- and thea2-globulin. He subsequently found (5), usingthe Anson hemoglobin assay for trypsin, that bothinhibitors react reversibly with trypsin, and thatthe dissociation constant for the inhibitor in thea,-globulin was 6 x 10-10 and that for the inhibi-tor in the a2-globulin, 3 x 10-1". Having foundthat urea makes the otherwise irreversible inhibi-tion of trypsin by soybean inhibitor appear to bereversible (5, 14), Jacobsson suggested that thereaction with serum trypsin inhibitors might beirreversible in the absence of urea.

The present report is concerned with somemodifications of the casein method of Kunitz (9)for the determination of tryptic activity, with thequestion of the reversibility of the reaction betweentrypsin and serum inhibitor, and with certain otherfactors bearing on the standardization of methodsfor the determination of trypsin inhibitor levelsin serum.

METHODSAND MATERIALS

Estimation of the concentration of trypsin and in-hibitor. Salt-free trypsin and soybean trypsin inhibitor(Worthington Biochemicals), twice crystallized andfive times crystallized, respectively, were used. Stocksolutions of trypsin and of soybean inhibitor were pre-pared in 0.0025 N HCl and the absorbance was deter-mined at 280 myA in a Beckman Model DU spectrophotom-eter. The factors 0.585 for trypsin and 1.10 for soybeaninhibitor, given by Kunitz (9), were used to convert ab-sorbance to mg. protein per ml. solution. Workingsolutions of both enzyme and inhibitor were prepared asindicated below.

Substrate. Three Gm. vitamin-free casein (Pfanstiehl)was suspended in 100 ml. of 0.17 M tris (hydroxymethyl)aminomethane brought to pH 7.6 with HCL. The sus-pension was heated in a boiling water bath for 15 min-

947

HALLIE F. BUNDYAND JOHN W. MEHL

,A TRYPSIN PERML. DIGESTION MIXTURE

FIG. 1. STANDARDIZATION OF TRYPSIN DIGESTION OF

CASEINThe absorbance of the supernatant from the trichloro-

acetic acid precipitate of the digest as compared to thetrypsin concentration: 0-0, in the absence of addedCa++; *--- , with Ca++ added to 4 X 103 M in the pre-incubation mixture before adding substrate.

utes to dissolve the casein. The solution had a final pHof 7.0.

Determination of trypsin activity. The casein digestionprocedure of Kunitz (9) was used with some modifica-tions. Since serum inhibitory activity disappears rapidlyon exposure to a pH of less than 6, all dilutions were

made with 0.17 M tris buffer, pH 7.6, rather than with0.0025 N HCOas in the original method of Kunitz. Aworking trypsin solution was prepared by diluting thestock solution 1: 10 with the buffer. To serial volumesof 0.1 to 0.8 ml. of the working trypsin solution, sufficientbuffer was added to bring the total volume in each tubeto 2 ml. These trypsin dilutions and the casein substratewere allowed to incubate separately for 15 minutes at350 C. in order to attain temperature equilibrium. Afterthis time 1 ml. of 3 per cent casein was added to eachof the trypsin dilutions and the mixtures were allowedto incubate at 35° C. Digestion was stopped after 20minutes by the addition of 6 ml. of 2.5 per cent trichloro-acetic acid. Blanks were prepared by adding the trichloro-acetic acid to the casein and then mixing with the trypsindilutions. It was found to be important to allow aboutone and a half hours after addition of trichloroacetic be-fore removing the precipitate by centrifugation. Theabsorbance of each supernatant was measured at 280 mAiwith a Beckman Model DU spectrophotometer. Absorb-ance, corrected for the blank value, was plotted againsttrypsin protein concentration as shown in Figure 1.

The original method of Kunitz employs a 0.5 per

cent casein concentration in the final digestion mixture.

It was found that the rate of reaction decreased during the20 minute digestion period, but that a linear rate couldbe maintained by increasing the final casein concentra-tion to 1 per cent. Under these circumstances the ratealso becomes linear with trypsin concentration over a

greater -range of trypsin concentrations.Using the- higher substrate concentration, some diffi-

culty was encountered with the release of digestion prod-ucts from the precipitate produced by adding 3 ml. of 5per cent trichloroacetic acid to the digestion mixture.More satisfactory results were obtained by using 6 ml.of 2.5 per cent trichloroacetic.

These changes, together with the change to preincu-bation at pH 7.6, have the disadvantage that our specificenzyme activities are not comparable with those obtainedby the original method. However, the reliability is im-proved by employing conditions in which zero orderkinetics are obtained. Specific enzyme activity is de-fined as the increase in absorbance per minute per lug. oftrypsin per ml.

Determination of inhibitor activity. A working soy-

bean inhibitor solution was prepared by diluting the stock

m

z

3EnDa

_0

bi 2ad

f-i

,U. SOYBEANINHIBITOR PERML.DIGESTION MIXTURE

FIG. 2. EFFECT OF CALCIUM ON THE INHIBITION OF

TRYPSIN BY SOYBEANINHIBITORResidual trypsin as a function of increasing amounts

of soybean inhibitor for two different trypsin concentra-tions: 0-O, in the absence of added Ca"+; 0---X, Ca++added to a concentration of 4 X 103 M in the preincuba-tion mixture before the addition of substrate.

948

SERUMTRYPSIN INHIBITOR

solution 1: 10 with the tris buffer. Serial volumes ofthis working inhibitor solution were added to a fixedvolume of the working trypsin solution and the final vol-umes adjusted to 2 ml. with tris buffer. Residual trypsinactivity was determined by adding 1 ml. of 3 per centcasein and completing the determination of activity de-scribed above.

In determining inhibition by serum, the serum was di-luted 1: 100 or 1: 200 with the tris buffer, and serialvolumes of these dilutions were added in place of the soy-bean inhibitor.

Blanks were prepared for the lowest and highest con-centration of serum used and intermediate blank valueswere calculated by interpolation. This correction wasnot required for soybean inhibitor, which contained noappreciable material absorbing at 280 my which was notprecipitated by trichloroacetic acid.

Additions of ethylenediaminetetracetate and calcium.Ethylenediaminetetracetate (EDTA) was added as thedisodium salt and calcium as CaCl2. The concentrationsindicated subsequently are those in the preincubation mix-ture, before the addition of substrate.

RESULTS

Inhibition of trypsin by soybean inhibitor

The effect of Ca++. Since Ca++ has been shownto increase the stability of trypsin at an alkaline pH(15, 16) and to increase the esterase and amidase

,activity of trypsin (17), it was decided to investi-gate the effect of this cation on the proteolysis ofcasein by trypsin and on the-observed combiningratio of trypsin and soybean inhibitor. Resultsare shown in Figure 1 and Figure 2. As indicatedin Figure 1, the addition of Ca++ to a concentrationof 4 x 10 -3M increases the specific activity of thetrypsin from 3.4 x 10-3 to 3.9 x 10-3. The inter-cept obtained by extrapolating the curves in Fig-ure 2 to zero free trypsin gives the amount of in-hibitor which should completely inhibit the totalamount of trypsin added. Combining ratios canbe calculated from these values. Combining ratiosobtained in this manner are CT/I 3 = 2.10 in theabsence of Ca++, and CT/I = 1.80 for Ca++ addedto 4 x 10-3 M. Thus Ca++ appears to have in-creased the activity of this sample of trypsin byabout 15 per cent and the observed combining ra-tio has decreased correspondingly.

Effect of ethylenediaminetetracetate (EDTA).The results of similar experiments showing the

8 CT/I = Micrograms of trypsin inhibited by 1 l.g. soy-bean inhibitor.

TABLE I

The effect of Ca++, ethylenediaminetetracetic acid andethylenediaminetetracetic acid plus Ca++ on

two trypsin samples and on their reac-tion with soybean inhibitor

Specificenzyme

Added Ca++ EDTA* activity CTtM/L. M/L. X103 I

Trypsin sample 'a'0 0 3.4 2.10

4 X 10-3 0 3.9 1.804 X 10-a 102 2.7 2.16

Trypsin sample 'b'0 0 3.2 2.28

5 X 10-6 0 3.410-3 0 3.7

4 X 10-3 0 3.7 1.88102 0 3.70 10-4 2.90 10-a 2.3 2.290 102 2.7

2.25 X 10-3 2.5 X 10-8 2.8 2.31

* Ethylenediaminetetracetic acid.t Micrograms of trypsin inhibited by 1 g. soybean in-

hibitor.

effects of Ca++, EDTA and Ca++ plus EDTA onthe specific activity and on the observed combiningratio with soybean inhibitor of two trypsin prepa-rations are summarized in Table I. The trypsinpreparations represent the same original crystal-line trypsin but since the experiments were per-formed two years apart, they are considered hereas different samples. Ca++ seems to exert a maxi-mumeffect at 10-3 M. In each case, the decreasein combining ratio parallels the increase in specificactivity. Low concentrations of EDTA decreasethe specific activity of trypsin in the absence ofadded Ca++. This may indicate the presence oftraces of Ca++ in the reagents, or of tightly boundCa++ or other cations in the trypsin molecule. Aswas observed by Green and Neurath (17), higherconcentrations of EDTAhave an activating effect.Although EDTA at lower concentrations maycause a 28 per cent decrease in trypsin activity,the combining ratio with soybean inhibitor re-mains unchanged. EDTAcompletely counteractsthe effect of added Ca+.

Table II summarizes the results of similar ex-periments performed with serum.4 The findings

4 The sera used in these experiments were samples ofpooled sera obtained from the Chemistry Laboratory ofthe Los Angeles County General Hospital.

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HALLIE F. BUNDYAND JOHN W. MEHL

were qualitatively similar to those obtained in ex-periments with soybean inhibitor.

The specific activities for both Tables I and IIwere calculated from trypsin concentrations basedon the absorbancy of the trypsin solutions. Val-ues of CT/I for Table I and of the amount oftrypsin apparently inhibited per ml. of serum(Table II) were calculated on the same basis.The corrected values for the inhibition of trypsinby serum, which are given in the last column ofTable II, were calculated on the assumption thatsoybean inhibitor combines with trypsin on anequimolar basis under the same conditions. Thisbasis for standardization will be considered inmore detail in the discussion.

Effect of preincubation time

Since it is known that trypsin undergoes autoly-sis in alkaline solution and that Ca++ increases thestability of trypsin at an alkaline pH (7, 8), ourresults indicated that Call was exerting a protec-tive action during the alkaline preincubation pe-riod. This possibility was investigated by deter-mining trypsin activity after varying periods ofpreincubation. Working trypsin solutions wereprepared by dilution of the stock solution with0.0025 N HCl or 0.0025 N HCl plus 4 x 10-3 MCa++. 1.8 ml. of the tris buffer or 1.8 ml. of thetris buffer plus 4 x 103 M Ca++ were allowed tocome to 350 C. and 0.2 ml. portions of the work-ing trypsin solution were added. At various inter-vals 1 ml. of 3 per cent casein was added and pro-

TABLE II

The effect of Ca++ and ethylenediaminetetracetic acid ontrypsin and its inhibition by serum

Trypsin inhibitedSpecific

Added enzyme Apparent CorrectedtCa++ EDTA* activity mg./ml. mg./m.M/L. MIL. X108 serum serum

Pooled serum sample A0 0 2.5 2.38 1.00

4 X 10-3 0 3.9 1.66 1.110 10-4 2.2 2.26 0.95

Pooled serum sample B0 0 2.9 2.61 1.24

4 X 10-' 0 3.9 1.90 1.270 10-3 2.1 2.64 1.26

* Ethylenediaminetetracetic acid.t Corrected on the basis of inhibition by soybean in-

hibitor under the same conditions (see text).

80-

R 20

12,STRYPSIN

6jugTRYfPSIN~~~c.~~~

0 5 10 15 20PREINCUBATIONT1ME,MIN.AT 35-C

FIG. 3. THE EFFECT OF PREINCUBATION OF PH 7.6 AND350 C. ON THEACTIVITY OF TRYPSIN

Expressed as the per cent of initial activity for 12 ug.trypsin: O-O, in the absence of added Ca++; @-- -, inthe presence of 4 X 10' M Ca++ in the preincubationmixture.

teolytic activity was determined by adding 0.2 ml.of the working trypsin solution to a mixture ofbuffer and casein. Subsequent activities were cal-culated as per cent of initial activity. Results areshown in Figure 3. As was expected, trypsin un-dergoes a loss of activity at pH 7.6. The initialrate of decay is quite rapid but gradually levelsoff. Ca++ does indeed afford partial protectionagainst this loss in activity. After 15 minutes pre-incubation at 350 C., about 70 per cent of the origi-nal activity remains in the absence of added Ca++,while 85 per cent remains when Ca++ is added.:

Dissociation of trypsin-serum inhibitor complex

The following experiments were carried outwith preincubation for 15 minutes at pH 7.6, inthe presence of 4 X 10-4 M Ca++. Under theseconditions, 1 mg. of crystalline soybean inhibitorwas found to inhibit 1.8 mg. of our trypsin prepa-ration. Since, as indicated in the discussion, 1mg. of soybean inhibitor should inhibit 1.2 mg. oftrypsin, apparent trypsin concentrations were cor-rected by a factor of 1.2/1.8.

The effect of increasing amounts of serum onthe tryptic proteolysis of casein is shown in Fig-ure 4a. Departure from stoichiometric inhibitionwith higher concentrations of serum indicates dis-

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-----o

SERUMTRYPSIN INHIBITOR

sociation of a trypsin-inhibitor complex. Thesame data are plotted in Figure 4b as per cent in-hibition by increasing amounts of serum, showingthat the inhibition is proportional to the amountof serum added up to about 70 per cent inhibition.This assumes significance in the determination ofserum trypsin inhibitor levels, since only valuesobtained at less than 70 per cent inhibition yield aproportionality between inhibition of activity andthe amount of inhibitor present.

Assuming the affinities of the two inhibitors inserum for trypsin are approximately the same, aswas found by Jacobsson, that 1 Mof each inhibitorcombines with 1 M of trypsin, and that the inhi-bition is noncompetitive,5 a dissociation constantwas calculated from the equation:

Kdis =FT FICT'

where FT and FI represent free trypsin and freeinhibitor, respectively, and CT represents com-bined trypsin. FT was obtained by reference tothe activity curve and CT by subtracting FT fromthe total trypsin added. If the assumptions enu-

5 This assumption may not be true in the strict senseof the word. However, a threefold decrease in substrateconcentration had no effect on the calculated Kdl..

11

merated above are correct, the ml. of serum ob-tained by extrapolation of the curves in Figure lacontain a total number of moles of inhibitor equalto the total moles of trypsin added. FI is thenobtained by subtracting CT from the total amountof inhibitor for each serum concentration. Allconcentrations were changed to moles per liter,using 24,000 as the molecular weight of trypsin(13). The dissociation constant calculated inthis manner has the value 8 x 10-1 M. In Figure5, the molar concentration of combined trypsin isplotted against the molar concentration of inhibitor.The solid line represents the theoretical curve cal-culated from the dissociation constant 8 X 10-10 M.

Serum trypsin inhibitor levels in normal persons

Serum trypsin inhibitor levels were determinedfor 40 apparently normal persons between the agesof 20 and 50 years. These were laboratory per-sonnel, medical students, and professional blooddonors. The mean for males was found to be1,025 and for females 1,037; therefore both sexeswere considered together. The mean was foundto be 1,032 jug. trypsin bound per ml. serum, witha standard deviation of plus or minus 127 and astandard error of the means of plus or minus 6.3pg. per ml. serum.

a

0 1 2 0 20 0 0 M1cMLSERUMPERMLDIE5STC MKIXTUREn10 %INHIINZUW

FIG. 4. INHIBITION OF TRYPSIN BY INCREASING CONCENTRATIONSOFHUMANSERUM

Three trypsin concentrations, plotted in 4a as free trypsin as compared to thequantity of serum; and the same data in 4b as per cent inhibition as a functionof the amount of serum added.

951

HALLIE F. BUNDYAND JOHN W. MEHL

x 76

hi

0

4-

I-4

0 i 5 6 7 8 0 1INHIBITOR, MOLESPERLITER X 108

FIG. 5. DIsSOCIATION OF TRYPSIN-SERUMINHIBITOR COMPOUNDThe points represent experimental values for combined trypsin as a function

of increasing total serum inhibitor. The curve is calculated assuming thattrypsin and serum inhibitor combine reversibly in equimolar quantities.

Analysis of the differences between duplicatedeterminations gave a standard deviation forsingle determination of plus or minus 1.6 per centand the standard deviation between duplicate anal-yses on different days was plus or minus 1.0 percent. At the mean normal value these would beplus or minus 16 and plus or minus 10 ug. trypsininhibited per ml. of serum. Since determinationswere always done in duplicate, the observed stand-ard deviation of plus or minus 127 Mug. from themean normal value is largely a reflection of thevariation within the population rather than of er-rors in the determinations.

DISCUSSION

The instability of trypsin above pH 5 compli-cates the determination of trypsin inhibitor in se-rum, since this inhibitor undergoes inactivation ata pH below 6. Although Ca++ stabilizes trypsin inalkaline solution, it does not completely preventthe loss of activity. Consequently, when trypsinis preincubated with serum inhibitor at pH 7.6 thetrypsin activity decreases as a result of inactivationas well as from combination with inhibitor.

It would be desirable to express inhibitor con-centrations in serum in terms of the amount of

trypsin inhibited per unit volume of serum. Inthe present study we have determined trypsinconcentrations by measuring the absorbance of thetrypsin stock solution at 280 mu, but such a meas-ure of trypsin concentration is subject to consider-able error because of the presence of protein im-purities, denatured trypsin, or trypsin autolysisproducts in the trypsin preparation. Kunitz (9)suggested that trypsin preparations be standard-ized by the reaction with soybean inhibitor. Ja-cobsson (5) has done this in his study of trypsinserum inhibitor. This basis for standardizationseems well justified, since highly purified soybeaninhibitor is commercially available and it has beenestablished that 1 Mof trypsin combines with 1 Mof inhibitor (18-20). Kunitz had originally takenthe weight combining ratio to be 1, but taking themolecular weight of soybean inhibitor to be 20,000(21), the theoretical value of CT/I would be 1.2.This is considerably lower than the lowest value,1.80, which we obtained (Table II). If the valuesof CT/I obtained with and without added Ca++are corrected by assuming that the loss of activityduring preincubation at pH 7.6 also represents lossof trypsin able to combine with inhibitor, the cor-rected values of CT/I become essentially the samein both cases, and average 1.63. This would sug-

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SERUMTRYPSIN INHIBITOR

gest that some 26 per cent of the absorbance ofour trypsin stock solution at 280 mu was due toinert material.

It might be argued that essentially the same re-

sults could be accomplished by standardizing re-

sults according to the specific activity of the tryp-sin. Assuming that both soybean and serum

inhibitor combine only with active trypsin, the re-

sults obtained with and without added calcium can

be harmonized in this way, but this method failsin the experiments with added EDTA. If we takethe behavior in the presence of Ca++ as a base,trypsin sample 'a' (Table I) has a specific activityof 3.9 x 10-3 and gives a value of 1.80 for CT/I.In the absence of added Ca++, the specific activityis 3.4 x 10-3 and gives a value of 2.10 for CT/I.In the absence of added Ca++, the specific activityis 3.4 x 10-3 or 13 per cent less. Taking this tomean that there is 13 per cent less trypsin than inthe presence of Ca++, the observed value of CT/Iof 2.10 should be reduced to 1.83, in good agree-

ment with the value of 1.80. However, in thepresence of EDTA, the specific activity is reducedby 31 per cent to 2.7. This would lead to a cor-

rection of the observed value of CT/I from 2.16to 1.50.. Similar results are obtained with serum

inhibitor. The results given in Table II, if cor-

rected for specific activity, would give the same

values for mg. trypsin per ml. serum for measure-

ments made with and without added Ca++, but a

different value in the presence of EDTA.The results obtained with serum, measured with

or without aded calcium or EDTA, do give thesame result if based on the amount of the trypsinavailable for combination with soybean inhibitorunder the same conditions. The corrected valuesgiven in the last column of Table II were obtainedby assuming that the value of CT/I for soybeaninhibitor is 1.2 under all conditions. For example,having an observed value of CT/I of 1.80 for soy-

bean inhibitor in the presence of Ca++, it was as-

sumed that the trypsin concentration was 1.20/1.80or 0.67 as great as indicated by the spectrophoto-metric determination on the stock trypsin solution.Consequently, rather than having 1.66 mg. oftrypsin inhibited by 1 ml. of serum, only 0.67 x

1.66, or 1.11 mg. was actually inhibited.The behavior in EDTA remains unexplained,

however. It would be logical to assume that thereduced specific activity in the presence of lower

concentrations of EDTAwas due to the sequester-ing of traces of Ca++ or other activating or stabiliz-ing ions. However, the effects on observed valuesof CT/I are not consistent with those obtained byadding Ca++.

The results which have been presented indicatethat the inhibition of trypsin by human serum isreversible and that although the dissociation isslight, it should be considered in the determinationof serum trypsin inhibitor levels. The value forthe dissociation constant is probably correct onlywithin an order of magnitude, since it was calcu-lated from very low residual trypsin activities.The calculation also involves certain assumptionswhich are probably not strictly true, namely thatboth inhibitors in serum have the same affinity fortrypsin and that the inhibition is noncompetitive.However, the satisfactory agreement between ob-served and theoretical results indicates that anydifference in the affinities of the two inhibitors fortrypsin is less than an order of magnitude and thatif the inhibition is competitive, it must be of thetype postulated by Green (22) for soybean trypsininhibitor and pancreatic trypsin inhibitor.

The average value of 1,030 ug. trypsin per ml.serum for the inhibitor level in normal, humanserum is somewhat higher than those reported byChristensen, 655, and by Jacobsson, 752, andlower than those obtained by Peacock and Sheehy,about 1,400, and by Shulman, 2,200. The lattertwo estimates failed to take into account the au-tolysis of trypsin during the alkaline preincubationperiod, which would contribute to the high valuesobtained. Christensen standardized his trypsinpreparation with soybean inhibitor. However inChristensen's method of assay, the highest dilutionof serum which allowed a clot to be formed in thepresence of trypsin, thrombin and fibrinogen wasassumed to contain a concentration of serum in-hibitor equivalent to the amount of trypsin usedin the test. Thus his measurements were madein the region of dissociation of the trypsin-inhibitorcomplex and would be expected to yield lowvalues. Jacobsson also standardized his trypsinwith soybean inhibitor using the casein digestionmethod, but his serum inhibitor determinationswere carried out with hemoglobin as the sub-strate. Using this procedure for soybean inhibi-tor, he reported an apparent combining ratio of1.54 Gm. trypsin per Gm. soybean inhibitor, rather

953

HALLIE F. BUNDYAND JOHN W. MEHL

than 1.85 when the casein substrate was used. Webelieve that the combination with soybean inhibitorshould be measured under precisely the same con-ditions as the serum measurements for which itwill serve as a standard. On this basis, and usinga theoretical value of 1.2 Gm. trypsin as we have,rather than the value of 1 Gm. trypsin per Gm.soybean inhibitor which was used by Jacobsson,his average value becomes 1,080. This is in goodagreement with our value of 1,032.

SUMMARY

The Kunitz casein method for the measurementof trypsin activity has been modified by increasingthe substrate concentration and changing the con-ditions for precipitation of undigested substrate(9). The modifications result in zero orderkinetics.

Using this assay procedure, the measurement oftrypsin inhibitor activity in serum has been exam-ined with respect to standardization. During therequired preincubation of inhibitor and trypsin atpH 7.6, there is a loss of trypsin activity which isinitially rapid, but becomes essentially zero after15 to 20 minutes. The presence of Ca++ decreasesthis rate of decay, but does not afford completeprotection.

The decrease in trypsin activity during prein-cubation produces an equivalent decrease in theamount of trypsin available for combination withboth soybean and serum trypsin inhibitors, asjudged by the behavior with or without addedCa++. In the presence of EDTA the specific en-zymatic activity of the trypsin may be further re-duced, but this does not result in a comparablereduction in the amount of trypsin apparently ableto combine with either serum or soybean inhibitor.

The results suggest that the soundest basis forstandardization of trypsin inhibitor values in se-rum is the assumption that the amount of trypsinavailable for binding is the same as that whichcombines with crystalline soybean inhibitor underthe same conditions that are employed for themeasurement of the serum inhibitor.

The inhibition of trypsin by human serum hasbeen shown to be reversible, and the significanceof this in the determination of serum trypsin in-hibitor levels has been pointed out. A dissociation

constant of 8 X 10-1 Mhas been calculated for thetrypsin-inhibitor complex. Certain assumptionsinvolved in the calculation have been discussed.

It has been found that 1 ml. of normal serumwill inhibit 1.03 ± 0.13 mg. of trypsin, and shownthat this can be harmonized with Jacobsson's re-sults. Reasons are presented for the differencesfrom other reported values.

REFERENCES

1. Camus, L., and Gley, E. Action du serum sanguinsur quelques ferments digestifs. C. R. Soc. Biol.(Paris) 1897, 4, 825.

2. Ascoli, M., and Bezzola, C. Das Verhalten des Anti-tryptischen Vermbgens des Blutserums bei dercroup6sen Pneumonie. Berl. klin. Wschr. 1903,40, 391.

3. Brieger, L.. and Trebing, J. Uber die antitryptischeKraft des menschlichen Blutserums, insbesonderebei Krebskranken. Berl. klin. Wschr. 1908, 45,1041.

4. Grob, D. The antiproteolytic activity of serum. I.The nature and experimental variation of theantiproteolytic activity of serum. J. gen. Physiol.1943, 26, 405.

5. Jacobsson, K. Studies on the trypsin and plasmininhibitors in human blood serum. Scand. J. clin.Lab. Invest. 1955, 7 (suppl. 14) 57.

6. Christensen, L. R. Methods for measuring the ac-tivity of components of the streptococcal fibrino-lytic system, and streptococcal desoxyribonuclease.J. clin. Invest. 1949, 28, 163.

7. Peacock, A. C., and Sheehy, J. J. Studies of varioustests for malignant neoplastic diseases. VII. Se-rum inhibitors of chymotrypsin and trypsin. J.nat. Cancer Inst. 1952, 12, 861.

8. Shulman, N. R. Studies on the inhibition of proteo-lytic enzymes by serum. II. Demonstration thatseparate proteolytic inhibitors exist in serum; theirdistinctive properties and the specificity of theiraction. J. exp. Med. 1952, 95, 593.

9. Kunitz, M. Crystalline soybean trypsin inhibitor.II. General properties. J. gen. Physiol. 1947, 30,291.

10. Hussey, R. G., and Northrop, J. H. A study of theequilibrium between the so called "antitrypsin" ofthe blood and trypsin. J. gen. Physiol. 1923, 5,335.

11. Shulman, N. R. Studies on the inhibition of proteo-lytic enzymes by serum. I. The mechanism of theinhibition of trypsin, plasmin, and chymotrypsinby serum using fibrin tagged with I"2 as a sub-strate. J. exp. Med. 1952, 95, 571.

12. McCann, S. F., and Laskowski, M. Determinationof trypsin inhibitor in blood plasma. J. biol. Chem.1953, 204, 147.

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SERUMTRYPSIN INHIBITOR

13. Jacobsson, K. Electrophoretic demonstration of twotrypsin inhibitors in human blood serum. Scand.J. clin. Lab. Invest. 1953, 5, 97.

14. Jacobsson, K. The effect of urea on the inhibition oftrypsin by soybean trypsin inhibitor. Biochim. etBiophys. Acta 1955, 16, 264.

15. Gorini, L. R6le du calcium dans le systeme trypsine-serumalbumine. Biochim. Biophys. Acta 1951, 7,318.

16. Bier, M., and Nord, F. F. On the mechanism ofenzyme action. XLVI. The effect of certain ionson crystalline trypsin and reinvestigation of itsisoelectric point. Arch. Biochem. 1951, 33, 320.

17. Green, N. M., and Neurath, H. The effect of di-valent cations on trypsin. J. biol. Chem. 1953,204, 379.

18. Kunitz, M. Isolation of a crystalline protein com-pound of trypsin and of soybean trypsin-inhibitor.J. gen. Physiol. 1947, 30, 311.

19. McLaren, A. Some observations on a trypsin tryp-sin-inhibitor system. C. R. Lab. Carlsberg, Serchim. 1952, 28, 175.

20. Steiner, R. F. Reversible association processes ofglobular proteins. VI. The combination of trypsinwith soybean inhibitor. Arch. Biochem. 1954, 49,71.

21. Cunningham, L., Tietze, F., Green, N., and Neurath,H. Molecular-kinetic properties of trypsin andrelated proteins. Disc. of Faraday Soc. 1953, 13,58.

22. Green, N. M. Competition among trypsin inhibi-tors. J. biol. Chem. 1953, 205, 535.

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