Salvatore F. Russo a n d Tom Holzman Western Washington State College Bellingham, 98225
The Activity of Trypsin
.~~~.
We a n exneriment entitled "The Turnover . .have ---.- adanted -~~~~~ Number of Trypsin and Chymotrypsin" found in "Experimental Methods in Modern Biochemistrv" hv G. Rendina ( 1 ) to illustrate the following points: ~ i r s t , t h a there t is a n imnortant difference in basina-enzyme concentration on weight, . Hbsorhance, or active sites. Second, that the method chosen to exnress enzvme concentration determines the calculated valu;of specific, molecular, and catalytic center activity.
Background The activity of a n enzyme is a n expression of the efficiency with which substrate is converted to product. Because varying iuteroretation of the term "turnover number" in expressing activity has led to confusion, it has been decided h y i n t e r n i tional agreement to recommend the discontinuance of this term. The specific, molecular, and catalytic center activity as defined by the Commission on Enzymes of the International Union of Biochemistry (2) are all based on the concept of a standard enzyme unit: One unit of any enzyme is defined as t.--h a t -amount will catalvze the transformation of one ~ - - ~ which ~ micromole of substrate per minute. All conditions such as temnerature. nH., substrate concentration. etc. should be stated. Specific activity is expressed a s units of enzyme per milliaam of nrotein. and should be calculated when the molecular weight of theenzyme is unknown. Molecular activity is defined as units oer micromole of enzyme which is equivalent to the number-of molecules of substrate transformed per minute per molecule of enzyme. When the enzyme has a catalytic center whose concentration can b e measured, the catalytic power can he expressed a s catalytic center activity: the number of substrate molecules transformed per minute per catalytic center. The enzyme trypsin catalyzes the cleavage of peptide honds wherever a carbonyl group is donated by lysine or arginine. Methylated casein is used as the substrate for the enzyme. The reagent, 2,4,6-trinitrobenzene sulfonic acid (TNBS) reacts with primary amino groups to form a substitution product a s shown below ~
~~~~~
,.
+
OiN -
SOH
+
H+
H NO, T h e derivative produced shows a n increased absorbance a t 340 nm compared with the reactants. Since t h e only primary amino groups we wish to measure are those formed by enzymatic cleavage, any preexisting primary amines must he blocked. This is accomplished by methylation of the basic amino acid residue side chains and the amino-terminal of the protein. The side chains are those of lysine (an €-amino) and arginine (a guanidino group). The guanidino group is not methylated nor does it react with T N B S because it is so strongly basic t h a t under the usual acylation conditions it is completely protonated. It has been found by Gorecki and 60 / Journal of Chemical Education
Shalitin (3) t h a t t r . w.s i n does not cleave a t c-N-N-dimethvllvsine residucs, most probably due to lark of hydrogen hond formation with the en7yme. Thus the only clea\,age points for trypsin will l,r ar thkarginine residues of the m&hylak?d sul,itrate. The new amino groups iormrd hy trypsin cleavage will he deterted hy t h e T N R S reaction. Experlmental Preparation and Dissolution of Methylated Casein Substrate For the preparation of themethylated casein substrate follow the procedure in the Instructors' Manual to Rendina's text ( 1 ) available from W. B. Saunders Company. Dissolve 1.5 g of Hammersten quality casein (Polysciences Inc.) in 150 ml of 0.1 M borate buffer, pH 9.0, and cool the mixture to 0% Stir the slightly cloudy solution and slowly add 300 mg of sodium borohydride. Add a few drops of 2-octanol to reduce foaming while stirring. Add 3 ml of 37%formaldehyde in 0.1-ml increments over a period of 30 min. Acidify the solution to pH 6.0 with 50% acetic aeid, and dialyze against several changes of deionized water. Lyophilize the desalted protein, label as methylated casein, and store at -20'C until needed. Do not dissolve the methylated casein in tris-(hydroxyrnethy1)aminomethane buffer as suggested in the Instructor's Manual to Rendina's text ( I ) since this is also detected by TNBS ( 4 ) .Prepare 100 ml of 0.1% (w/v) methylated casein in 0.05 M borate buffer, 5.75 X 10VM CaC12, at pH 8.1. Purification of TNBS
The TNBS commercially available is often not pure enough to give accurate optical determinations of new amino-terminals. Its molar absorptivity at 340 nm when suitahlypure will be less than 600 M-' cm-' (5). Using the di or trihydrate facilitatesthe purification process. One part TNBS (1-25 g) is dissolved with heating in an equal part by weight (1-25 g) of water, and concentrated hydrochloric aeid is added to make the solution 2 M in acid. The product precipitates on cooling and appears as a fine whitecrystalline solid. After one or tworecrystallizations the suitably pure TNBS will have a melting point of at least 170°C although the reported value is 185'C (5). Since the oxidation of sulfite is catalyzed by metals, solutions should be made up in distilled deionized water. We store this product in a vacuum desiccator to minimize the possibility of absorption of atmospheric water. Preparation of Trypsin Stock Solution Preoare the stock solution of trwsin .. in 10W M HCl with an aoproximate concentration of 1 mglml based on weight. This will be directly suitable for the active site titration. The procedure involving the tryptic cleavage of meth~latedcasein will require approximately a 50-fold dilution of this stock solution. Concentrationof Trypsin Based on Absorbance Determine the absorhanee s t 280 nm of an appropriate trypsin solution. Usine.. a soeeific absorotivitv . of 1.44 em-' ml/e 16) calculate the conrmrmtlnn in units of mg ml based on nhsorhanre measuremmLi. Assurnmg a mdecular weigh! of2R.R00rnlrulnte the cmcrnlrarion of mltn uf umdes ml based on ahsorhnnre mea*urementa.
.
.
Trypsin-Catalyzed Hydrolysis of Methylated Casein Follow the procedure in Rendina's laboratory manual ( I )
(a) To suitable test tubes add 0.1 ml of trypsin solution containing 1pg of enzyme to 1 ml of 0.1% (wlv) methylated casein,pH 8.1. Incubate the mixture for 30 min at 37%. Do this in triplicate. (b) The blank without enzyme is prepared by adding 0.1 ml of H20 to 1ml of substrate. (e) Stop the reaction by immersing the samples briefly in a boiling
water bath. Care must be taken to inactivate trypsin without causing the coagulation of methylated casein. (d) Add, consecutively, 1ml of freshly prepared 0.1% TNBS and 1 ml of 4% NaHCOa, pH 8.5. Incubate these mixtures in the tubes for 30 min at 50% in the dark. (e) After this treatment add 1 ml of 10%sodium dodecyl sulfate and 0.5 ml of 1M HCI to each tuhe. (f) Read the absorbance s t 340 using the blank as a reference. Increase the wavelength up to 380 nm if it is not possible to zero the speetrophotometer at 340 nm. Active Site Titration Follow the general procedure of Chase and Shaw (7). Using I-cm cells in a double beam recording spectrophotometer,place 1.00 ml of the trypsin staek solution and 0.975 ml of 0.1 M vemnal-0.02 M CaClz at pH 8.3 in the sample cell. Place 1.975ml of the same huffer in the reference cell and balance the instrument. Add 0.025 ml of 0.01 M p-nitrophenyl p'-guanidinobenzoate HC1 (NPGB) dissolved in dimethyl formamide to the reference cell using a 2 5 4 micropipet. Add 25 pl of the NPGB solution to the sample cell, mix thecontents, and follow the absorbance at 410 nm as a function of time.
Table 1.
Concentration of'Trypsin
Concentrafion Concentration concentration Based o n Weight
Stock Tryorin concentration
1.06
,mn,m,, ~ , Concentration used 2,
tn rnethylated
2.12 X 1 0 4
1.28
x 10-b.932x
8.91 X
5.38
x
c a r e r array lm910.1 mll
cdniintration urea in methylatea
Table 2.
10"
A340 (0.00461) = liters (1 cm) 104 mole em male of WNHZformed) A:uo(0.35 X = A:~d0.35pmale of n-NH2 formed) (AX4")(0.35 pmole a-NH2 formed) 30 min Specific activity = mg of enzyme
----)
( A : ~ ) ( 0 . 3pmole 5 n-NHz formed) 30 min
Active Site Titration Extrapolate the curve 0fA4~0versus time hack to zero time. Calculate the active enzyme concentration from Beer's law using a molar ahsorptivity of 16,595 M-' cm-I for p-nitrophenol a t p H 8.3 a s follows Am (16,595 M-'cm-')(I cm) A complete discussion of the active site titration and the acyl enzyme mechanism has been described in this Journal (8). [active trypsin] =
Results and Discussion Table 1 shows t h a t a stock solution of trvwsin with a cal-
ConcenrraConcenfra
SpecificActivity (gmole ruartratel r n l n l m g enzyme) Molecular Activity lumole rubrtrate/min/ . . ,hole enzyme) Catalytic c e n t e r ~ctivity (prnole rubrtratelminl pmole active rite)
x lo-'
f i o n Based on Absorb-
Concenrra~ fion Bared on Abrorb~
bance
ance
1.67
2.76
...
39.7
65.8
...
...
.. .
90.1
decreases t o 0.466 melml when hased on the active site titration. This further ccange occurs because the ahsorhance a t 280 nm measures both active and inactive enzvme whereas the active site titration is specific for active enzyme only. T h e active site titration takes advantage of the fact that a t early rlmei in the reaction thecnnceutmtion ofp.nirrophenol liberated can he relaredmi a 1:l basis with rhe roncenrmtim of active trypsin in solution. Also included in Tahle 1 are comparisons of concentrations used in the methylated casein assay hoth in units of mgl0.l ml and .umolesl0.1 ml. Tahle 1shows n. that there is an important difference in basing enzyme concentration on weight, ahsorhance, or active sites. Tahle 2 shows the results for the calculation of the activity of trypsin. T h e specific activity has a value of 1.67 when the concentration is hased on weight and increases to 2.76 when hased on ahsorhance. The molecular activity likewise changes from 39.7 when hased on weight to a value of 65.8 when hased on ahsorhance. T h e increase of approximately 65% in specific activity or molecular activity is therefore attributable t o the method chosen to determine enzyme concentration. T h e catalytic center activity can only he determined from an active site titration and was determined t o be 90.1. This value is similar to one which can be calculated from Figure 2 in the article by Lin, Means, and Feeney (4). Tahle 2 shows that the calculation of activity is dependent upon the method of determining concentration. ~
~
~
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Literature Cited I l l Rendina. G., "Experimental Methods in Modern Biochemistry." W. 8. Ssunderi Cu.. ~hiladelphia,1971.g. 215.
121 "Report ofthe Commision on Enzymes ofthe InUrnafinnsl Union of Riorhemisfry."
oxford.perearnonpress.,961.
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fact that the lyophilized protein contains salts and absorbed water which to the weight hut not the This table also shows that the calculated concentration further
3.92
10"
10.'
Activity of Tryprin Bared on W e i g h t
)
tivesite 0.466
0.640
-
tion
Trypsin-Catalyzed Hydrolysis of Methylated Casein The average value of molar ahsorptivity for various trinitrophenyl amino acid derivations is 1.3 X lo4MM-'em-' a t 340 nm (4). Using this figure it is possible t o calculate the concentration of liberated amino groups which is, in turn, equivalent t o substrate bonds cleaved. Therefore, making use of Beer's law A340 c=-Am liters (1.3 x KI(1cm) mole cm Since the total volume is 4.6 ml, the total moles of a-NH2 formed is
Based on Ac-
Titration
Calculations
Molecular activitv =
Bared on
Absorbance
.
.... . . .
. ... ...... ...~... ",..
. . ..... , .. ...
.,
.
...
~ 1 ~~~d~~~~ . press. !ip YWI, WII. rZSR. I,. 16s. 161 Davie. E. W., andNeurath, H., J. B i d Chem., 212,507 119551.
171 Chase,.lr.,T.,sndShsw, E..Biochem. Riaphys RPI. Cemmiin., 29.508 119671. (8) ~ e n d e r M. . L..~ k d yF. . ~..snd wedier. F.c..J.CHEM. ~~uc.41.84. 119671.
Volume 54, Number 1, January 1977 / 61
