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Assay of proteolytic enzyme activity using a 14C-labeled hemoglobin
ANALPTICAL
BIOCIIEMISTRY
Assay
42, 214-221 (1971)
of Proteolytic
Enzyme
a 1-4C-Labeled ,J,41- S. ROTH Section
Activity
Using
Hemoglobin THOMAS
AND
of Biochemistry and Biophysics, ~~triuersily of Conwcfimt, Storm.
LOSTY
Biologicnl
Sciences
Cotlnecficut
/X%3
Group,
AND
EUGEN Food
Labornfory,
U. S. Army
Nalick
Recrivrd
WIERBICKI Laboratories, November
NIltick,
Masmchusetts
01760
17, 1970
Several years experience in our laboratories has shown that, present standard techniques (l-4) for assay of total proteolytic activity are unsatisfactory from several points of view. In particular, in many tissues with low activity, results are poorly reproducible, long incubation times are required, and blanks arc, in general, so high that the reliability of values obt,ained is open t,o considerable quest.ion. Oft’en the tissue itself is the preferred substrate and addit’ion of anot,her substrate, such as hemoglobin, may competitively depressproteolytic activit,y, resulting in blanks which gave higher readings than the experimental assays. These difficulties can bc largely overcome by use of a substrate that contains a radioactivity labeled amino acid which will be released as prot~eolytic activky cont,inues. In this study hemoglobin labeled with KWNO was used as the substrate. The amount of radioactivity appearing in the acid-soluble fraction after precipitation of t#heprotein will be a measure of ln-oteolytic activity on the labeled substrate and any endogenous release of amino acids will not interfere since these amino acids will not be labeled. Furthermore , sensitivity should be great’ly improved as radioact’ivity can hc dctectcd at, lcvcls much lower than for calorimetric or spect~ropliotonictric assays. Optimal conditions for the labeling, some properties of the labeled substrate, and it’s use in assay for total protcolytic activity of various aamplcs are discussed in this paper. 214
Beef hemoglobin (Sigma Chcmirnl c’o., type T! 76B-1620) : 400 mg, was tlissolved in 15 ml of gla.+distillecl nntcr and tlrc pH adjust’ed to 6.1 with 0.1 JV NaOH. The volume was 1)rought to 20 ml and 0.4 ml 10.04 mCi) of IP4CN0 was added. The mixture was incubated at 50” for 2 hr and then allowed to stand overnight in the cold room. In the morning 2 ml (20 qolcs) of cysteine hydrochloride in water (adjusted to pH 6.1) was added and the sample incubated at, 37” for 2 hr. The solution was dialyzed against several changes of clistillcd water for 48 hr in t#hc cold. The specific activity of the hemoglobin with t#he amount of radioactivity used is in the range 2500 to 3500 q)~l~//rng. It can be increased by increasing the all~ount of K’“CNO. 2. Assa!/
of Tissue Samples-Standnrd
Assay
Duplicate test tubes arc set “1) for each sample containing 1.4 ml of 0.2 N acetate buffer, pH 3.8, 0.7 ml of enzyme solution (with muscle and liver, 440 mg/ml or less is used’), and 1.0 ml of sub&ate containing 16 mg of hemoglobin. The rnixturc is incubated in a shaking water bath at 37”, the test t’ubes being cork1 to prevent evaporation. Aliquotx are removed at, desired intervals, the intcrvnl depending on the activity of the sample. Generally, 0.85 ml is rcmovetl ancl precipitated wit,11 0.2 ml of ice-cold 50% triclilorowcctic, acid and the mixture is agitated on a Vortex mixer. The tubrs are ccntrifugecl in a refrigcratcd Intcrnntionnl PR2 centrifuge at, 2000 rpm for 30 mm, and that supernat’ant fract’ion filtered through glass wool to remove any l)articles that might adhere to t’he meniscus. Then 0.1 ml is added to 15 ml of Bray solution and counted to the 95% confidence limit in a Nuclear-Chicago liquid scintillation counter. Appropriatc substrate blanks are run by incubating substrate and adding tissue after the precipitating agent. has been added. Tissue blanks are not required.
1. Factors
Affecting
Labeling
200 mg samples of hemoglobin were dissolved in 7.0 ml of glass-distilled water; the pH was adjusted to the desired value, Ii14CN0 added to each sample, and the volume made up to 10 ml with glass-distilled water. The
216
ROTH,
LOSTY,
AXD
WIERBICKI
Pptd. miri Counts,/4
PH Ilxpk 6.1
ml
I 2590 “67!) ‘2054 I . ‘07’; 1446 1539 1085 1092 732 75.5
6.6 7.1 7 .6 8.1 Expt. ” 6.1 5 6 5.1 4.6 4.1 Experimental
min/O.l
samples, c~)untsj’4 ill sup!. fraction (0.1 ml)
det,ails
49s 429 .510 4!)H 4%1 470 345 3.55 420 389
3690 4370 4010 4070 3340 are described
in the text.
It is clear from Table 1 that the pH of labeling is an important factor in the final radioactivity of the hemoglobin sample prepared with the conditions described. On the other hand, the blank value is not appreciably affected by the pH of labeling so the extra radioactivity bound at the lower pH values is firmly bound and uot rcmorablc by acid precipitation. In the second experiment which employed lower pH values (Table 1j maximum labeling was obtained at pH 5.6 but the labeling at pH 6.1, 5.1, and 4.6 was only slightly less. (b)
Sfnbilif;/
of IAbel to Ijiffe~renf
Trenfnzents
The labeling procedure is patterned after that of Stark et (~1.C.5j and gives carbamyl derivatirea of free amino group:: (terminal or lysine) . It is possible t’hat other reactions may take place and it appears likely that the heme may bind cyanate as well. (SW reaction with cysteine on page 217.) Hemoglobin labeled as described under “Methods” is quite stable t’o
storage. It may be kel)t indefinitely at -20” without any increase in the blank values. Incubation at 37” for 25 hr in acetate buffer, pH 3.8, also does not significantly affect the label as no increase in the acid-soluble material occurs under these conditions. The label apl>ears to be stable at higher pH wlues a?: wvcll. Tnrulxttion at 37” for 1 hr in I)hosI)hate buffer, I~H 7.8, did not inrrc:w thus amount of acicl-soluble blank. Arginase, either without or with the addition of a Iwoteolytic enzyme, :A0 had no effect on the substrutv or the rwtc of release of acid-soluble radioactivity. Effect of cysteine ant/ oflrer srclfhytlryl co~~poz~ntls. 11%en cysteine hydrochloride is adjusted to l)H 3.8 and added to substrate labeled witho,& the use of cystcine, a ral)itl release of radioartirity to the acid-soluble filtrate occurs, a:: illuatrattd in Fig. I. The rate of liberation increased with incwasing conwntration of cyst&w. With the higher concentration, :L 1)lateau war rcachcvl at al)out 1 hr, after which thcrc was little further inrreaw. Greater amounts of rpstcinc than I mg/‘tubc (lid not release more activity but8 the I>latclau was reached sooner. Although there arc several way ‘s of explaining the action of cysteine the most likely one ia that cywteine is complexed to heme more firmly than cyanatc and releaw the latter. The reaction is competitive so higher concentrations of cysteine release the labeled cpanate more raI)idly. gi1.e idcwticnl results to ~It,rcxI)to~tll:lnol and reduced glutnthione qvstciiw.
I 0
15
I 30 MINUTES
45
I 60
218
ROTH,
LOSE,
AND
WIERDlCKI
By treatment of the labeled substrat’e with an excess of cysteine as described under “Methods,” the heme-bound cyanate is released and the hemoglobin then becomes insensitive to possible variations in the sulfhydry1 content of homogenates or other enzyme preparations. Amount of radioactivity precipitated. Under standard incubation condit,ions a minimum of 95% of the radioactivity contained in the hemoglobin is precipitated by TCA. The radioactivity not, precipitated may represent a small amount of hydrolysis of the protein or of the carbamyl groups by the acid. Effect of different concentrations of li?TXO on. labeling of cysteinebated hemoglobin. 1 gm of hemoglobin was dissolved in 40 ml of glassdistilled water, adjusted to pH 6.1 and to 50 ml, and divided into five 10 ml portions. Each portion was then t,reatecl with a different amount of K14CN0, followed by cysteine as described under “Methods.” The results are given in Table 2. Efl’ec(
of I)ifferetti, .4mt.
Amounts
of K’CNO
on Labelittg
of KWNO, mCi
Hemoglohirt sp. act ., cpmirng
0, 08
“75 (X62 1 I!i,:iB:: 50 ( 700 ‘22 ( .x%i 10,572
0 04 0.02 0.01 0,005 The
experimental
procedure
of Hemoglot)itt
is described
in the text.
The data in Table 2 show that, the amount of labeling with these condit,ions is approximately proportional to t,he concentration of K14CN0. The greater specific activit’y in this experiment compared to the standard procedure described under “Methods ” is probably due to t’he higher rat,io of radioactivit,y to hemoglobin. It is possible that by using greater amounts of label higher sensitivity can be achievetl. No extensive experiments were run to determine this. Effect of temperature on labeling. Incubation of hemoglobin with K14CN0 at 23”, 50”, or 60” gave no differences in the degree of labeling. Substrates labeled at the three temperatures when tested against the same liver and musck homogcnat,e all yic>l(led the rame results. The standard labeling t8cnipt~rature ;ItLvl)tc~~l,tllcrc~forc~,was room tc~lnprraturr. APPI,ICABILITY
OF
THIC
MIC’I’HOI~
7.2
IS;“1 I -
SI
1417 1775 17!l!I 1 (iti: 2ot2
IOS 7:; 54 ici
57
!I8
300 gm male Holtzman rat. Separwtc portions were tested using 5 different samples of cyanate labeled hemoglobin all prepared at the same time. A sixth sample of muscle was used with a mixture of all 5 hemoglobin substrates. The results arc given in Table 3 and they indicate that all five preparations of hemoglobin gave essent,ially the same activit,y for the muscle homogenate. USC of the mixture gave a somewhat higher value but the difference may not bc significant. Linearity of enzyme reaction with respect to tinle and enzyme a?nount. With rat liver or muscle homogenate, the production of acid-soluble radioactivity is linear for at least 4 hr (Figs. 2 and 3). The proportionality between enzyme amount and activit’y, however, is maint,ained only for lower amounts of enzyme. When very small amounts of proteolytic activ-
i 3 8
8,000
Y :
6,000
i
Substrate
TIME,
rut
blank
HOURS
FIG. 2. Relation bctwwn rnzymr~ amomlt and wtirity. Standard liver homogenate. (Amounts of tissue per ml homogenate.)
nssnq- proc~edur,~
:
220
ROTH,
LUSTY,
AND
-----------------
I
I I
U’IERBICKI
Substrate
/
1
I
2
3
4
TIME.
rat
FIG. 3. Kclation gkrocnrmus
ity are measured more.
between enzyme muscle homogenate.
the linearity
blank
amount, and (Amounts
HOURS
actirit,y. Standard of &sue per ml
of the reaction
assay procedure homogenate.)
may continue
;
for 24 hr or
DISCUSSION
The use of ‘“C-labeled hemoglobin as a substrate for determining total proteolytic activity offs 1rLv several advantages over present methods. The trchnique of labeling is simple and the substrate is stable and may be stored for long periods of time. Over t,no years’ experience in our laborat,ory wit’h this substrate embodying thousands of assays has demonstrated that the method is reproducible, quick, and rcliablc. In addition, we have used it to assay samples with prot’colytic activity so low that it is doubtful that it could have been detected by other mct~hotls. It is important to point out that this method, as others which use an added substrate, does not measure autolysis. It is clear from experiments carried out in our laboratory that proteolytic autolysis is inhibited by addition of an additional protein substrate, and what one is measuring is the ability of the tissues to degrade this added sub~trwtc to acid-soluble products. It is possible that the method may be applied to other special substrates, either natural or synthetic, with the same or other advantages. \T’e have tried, however, 14C-labeled lysozymc. which we found to be
The ineasurenient of total tiww proteolyt~ic :ivtivitp with :L hemoglobin substrate labeled with K1’CSO is dcwribcd. The labeled hemoglobin is stable and ik use offers sereral advanltnge~~ particularly in qwificGt,y and sensitivit,y owr incthods c*omi~onlp in me.
This work has been supported by the U. 8. Army Natick Laboratories undts1 contracts D$ IO-12%AMC-594 (S) anri I>&~(:-17-67-C-0156 with the Universit! of Connecticut. Jay Y. Roth was the l~rin(iI)al in\-estigator and Eugrn Wicrbicki, Chief, Irradiated Food Products Division, Food J&oratory, U. 8. Army Natirk Laboratories, was the Projwt OlIiwr for the subject research contracts. J. S. Roth is also a recipient of Curwr A\v:ud RC 63-13 from the Xationnl Instituiw of Health. S:ll ional Cancer Instit,ute.
1. 2. 3.
hl. I,.. J. (;etc. I’h&Yid. 22, 8 (l%s). H. I%, .4ND DIMLIS, J. T.. ,!&ci~r~. d. 87, 403 (1963). DE DUVE, C., PRESSMAN, B. C., GIAN~TO, R., Wa~~~arrx, R., AND AWLFZIINS, Uiochra. J. 60, 604 (1955). 4. PI,.\sT.~. R. J., .IND (;RUBICR, >I., BMLZ. Liiochen~. 5, 360 (1963). 5. %r.mli. G. I<.! STEIN, m-. H., AND RIOORE, S., J. Bid. (Ihem. 235, 3177 (1960). ;1NSON.
FELL,
I;.,
BIOCIIEMISTRY
Assay
42, 214-221 (1971)
of Proteolytic
Enzyme
a 1-4C-Labeled ,J,41- S. ROTH Section
Activity
Using
Hemoglobin THOMAS
AND
of Biochemistry and Biophysics, ~~triuersily of Conwcfimt, Storm.
LOSTY
Biologicnl
Sciences
Cotlnecficut
/X%3
Group,
AND
EUGEN Food
Labornfory,
U. S. Army
Nalick
Recrivrd
WIERBICKI Laboratories, November
NIltick,
Masmchusetts
01760
17, 1970
Several years experience in our laboratories has shown that, present standard techniques (l-4) for assay of total proteolytic activity are unsatisfactory from several points of view. In particular, in many tissues with low activity, results are poorly reproducible, long incubation times are required, and blanks arc, in general, so high that the reliability of values obt,ained is open t,o considerable quest.ion. Oft’en the tissue itself is the preferred substrate and addit’ion of anot,her substrate, such as hemoglobin, may competitively depressproteolytic activit,y, resulting in blanks which gave higher readings than the experimental assays. These difficulties can bc largely overcome by use of a substrate that contains a radioactivity labeled amino acid which will be released as prot~eolytic activky cont,inues. In this study hemoglobin labeled with KWNO was used as the substrate. The amount of radioactivity appearing in the acid-soluble fraction after precipitation of t#heprotein will be a measure of ln-oteolytic activity on the labeled substrate and any endogenous release of amino acids will not interfere since these amino acids will not be labeled. Furthermore , sensitivity should be great’ly improved as radioact’ivity can hc dctectcd at, lcvcls much lower than for calorimetric or spect~ropliotonictric assays. Optimal conditions for the labeling, some properties of the labeled substrate, and it’s use in assay for total protcolytic activity of various aamplcs are discussed in this paper. 214
Beef hemoglobin (Sigma Chcmirnl c’o., type T! 76B-1620) : 400 mg, was tlissolved in 15 ml of gla.+distillecl nntcr and tlrc pH adjust’ed to 6.1 with 0.1 JV NaOH. The volume was 1)rought to 20 ml and 0.4 ml 10.04 mCi) of IP4CN0 was added. The mixture was incubated at 50” for 2 hr and then allowed to stand overnight in the cold room. In the morning 2 ml (20 qolcs) of cysteine hydrochloride in water (adjusted to pH 6.1) was added and the sample incubated at, 37” for 2 hr. The solution was dialyzed against several changes of clistillcd water for 48 hr in t#hc cold. The specific activity of the hemoglobin with t#he amount of radioactivity used is in the range 2500 to 3500 q)~l~//rng. It can be increased by increasing the all~ount of K’“CNO. 2. Assa!/
of Tissue Samples-Standnrd
Assay
Duplicate test tubes arc set “1) for each sample containing 1.4 ml of 0.2 N acetate buffer, pH 3.8, 0.7 ml of enzyme solution (with muscle and liver, 440 mg/ml or less is used’), and 1.0 ml of sub&ate containing 16 mg of hemoglobin. The rnixturc is incubated in a shaking water bath at 37”, the test t’ubes being cork1 to prevent evaporation. Aliquotx are removed at, desired intervals, the intcrvnl depending on the activity of the sample. Generally, 0.85 ml is rcmovetl ancl precipitated wit,11 0.2 ml of ice-cold 50% triclilorowcctic, acid and the mixture is agitated on a Vortex mixer. The tubrs are ccntrifugecl in a refrigcratcd Intcrnntionnl PR2 centrifuge at, 2000 rpm for 30 mm, and that supernat’ant fract’ion filtered through glass wool to remove any l)articles that might adhere to t’he meniscus. Then 0.1 ml is added to 15 ml of Bray solution and counted to the 95% confidence limit in a Nuclear-Chicago liquid scintillation counter. Appropriatc substrate blanks are run by incubating substrate and adding tissue after the precipitating agent. has been added. Tissue blanks are not required.
1. Factors
Affecting
Labeling
200 mg samples of hemoglobin were dissolved in 7.0 ml of glass-distilled water; the pH was adjusted to the desired value, Ii14CN0 added to each sample, and the volume made up to 10 ml with glass-distilled water. The
216
ROTH,
LOSTY,
AXD
WIERBICKI
Pptd. miri Counts,/4
PH Ilxpk 6.1
ml
I 2590 “67!) ‘2054 I . ‘07’; 1446 1539 1085 1092 732 75.5
6.6 7.1 7 .6 8.1 Expt. ” 6.1 5 6 5.1 4.6 4.1 Experimental
min/O.l
samples, c~)untsj’4 ill sup!. fraction (0.1 ml)
det,ails
49s 429 .510 4!)H 4%1 470 345 3.55 420 389
3690 4370 4010 4070 3340 are described
in the text.
It is clear from Table 1 that the pH of labeling is an important factor in the final radioactivity of the hemoglobin sample prepared with the conditions described. On the other hand, the blank value is not appreciably affected by the pH of labeling so the extra radioactivity bound at the lower pH values is firmly bound and uot rcmorablc by acid precipitation. In the second experiment which employed lower pH values (Table 1j maximum labeling was obtained at pH 5.6 but the labeling at pH 6.1, 5.1, and 4.6 was only slightly less. (b)
Sfnbilif;/
of IAbel to Ijiffe~renf
Trenfnzents
The labeling procedure is patterned after that of Stark et (~1.C.5j and gives carbamyl derivatirea of free amino group:: (terminal or lysine) . It is possible t’hat other reactions may take place and it appears likely that the heme may bind cyanate as well. (SW reaction with cysteine on page 217.) Hemoglobin labeled as described under “Methods” is quite stable t’o
storage. It may be kel)t indefinitely at -20” without any increase in the blank values. Incubation at 37” for 25 hr in acetate buffer, pH 3.8, also does not significantly affect the label as no increase in the acid-soluble material occurs under these conditions. The label apl>ears to be stable at higher pH wlues a?: wvcll. Tnrulxttion at 37” for 1 hr in I)hosI)hate buffer, I~H 7.8, did not inrrc:w thus amount of acicl-soluble blank. Arginase, either without or with the addition of a Iwoteolytic enzyme, :A0 had no effect on the substrutv or the rwtc of release of acid-soluble radioactivity. Effect of cysteine ant/ oflrer srclfhytlryl co~~poz~ntls. 11%en cysteine hydrochloride is adjusted to l)H 3.8 and added to substrate labeled witho,& the use of cystcine, a ral)itl release of radioartirity to the acid-soluble filtrate occurs, a:: illuatrattd in Fig. I. The rate of liberation increased with incwasing conwntration of cyst&w. With the higher concentration, :L 1)lateau war rcachcvl at al)out 1 hr, after which thcrc was little further inrreaw. Greater amounts of rpstcinc than I mg/‘tubc (lid not release more activity but8 the I>latclau was reached sooner. Although there arc several way ‘s of explaining the action of cysteine the most likely one ia that cywteine is complexed to heme more firmly than cyanatc and releaw the latter. The reaction is competitive so higher concentrations of cysteine release the labeled cpanate more raI)idly. gi1.e idcwticnl results to ~It,rcxI)to~tll:lnol and reduced glutnthione qvstciiw.
I 0
15
I 30 MINUTES
45
I 60
218
ROTH,
LOSE,
AND
WIERDlCKI
By treatment of the labeled substrat’e with an excess of cysteine as described under “Methods,” the heme-bound cyanate is released and the hemoglobin then becomes insensitive to possible variations in the sulfhydry1 content of homogenates or other enzyme preparations. Amount of radioactivity precipitated. Under standard incubation condit,ions a minimum of 95% of the radioactivity contained in the hemoglobin is precipitated by TCA. The radioactivity not, precipitated may represent a small amount of hydrolysis of the protein or of the carbamyl groups by the acid. Effect of different concentrations of li?TXO on. labeling of cysteinebated hemoglobin. 1 gm of hemoglobin was dissolved in 40 ml of glassdistilled water, adjusted to pH 6.1 and to 50 ml, and divided into five 10 ml portions. Each portion was then t,reatecl with a different amount of K14CN0, followed by cysteine as described under “Methods.” The results are given in Table 2. Efl’ec(
of I)ifferetti, .4mt.
Amounts
of K’CNO
on Labelittg
of KWNO, mCi
Hemoglohirt sp. act ., cpmirng
0, 08
“75 (X62 1 I!i,:iB:: 50 ( 700 ‘22 ( .x%i 10,572
0 04 0.02 0.01 0,005 The
experimental
procedure
of Hemoglot)itt
is described
in the text.
The data in Table 2 show that, the amount of labeling with these condit,ions is approximately proportional to t,he concentration of K14CN0. The greater specific activit’y in this experiment compared to the standard procedure described under “Methods ” is probably due to t’he higher rat,io of radioactivit,y to hemoglobin. It is possible that by using greater amounts of label higher sensitivity can be achievetl. No extensive experiments were run to determine this. Effect of temperature on labeling. Incubation of hemoglobin with K14CN0 at 23”, 50”, or 60” gave no differences in the degree of labeling. Substrates labeled at the three temperatures when tested against the same liver and musck homogcnat,e all yic>l(led the rame results. The standard labeling t8cnipt~rature ;ItLvl)tc~~l,tllcrc~forc~,was room tc~lnprraturr. APPI,ICABILITY
OF
THIC
MIC’I’HOI~
7.2
IS;“1 I -
SI
1417 1775 17!l!I 1 (iti: 2ot2
IOS 7:; 54 ici
57
!I8
300 gm male Holtzman rat. Separwtc portions were tested using 5 different samples of cyanate labeled hemoglobin all prepared at the same time. A sixth sample of muscle was used with a mixture of all 5 hemoglobin substrates. The results arc given in Table 3 and they indicate that all five preparations of hemoglobin gave essent,ially the same activit,y for the muscle homogenate. USC of the mixture gave a somewhat higher value but the difference may not bc significant. Linearity of enzyme reaction with respect to tinle and enzyme a?nount. With rat liver or muscle homogenate, the production of acid-soluble radioactivity is linear for at least 4 hr (Figs. 2 and 3). The proportionality between enzyme amount and activit’y, however, is maint,ained only for lower amounts of enzyme. When very small amounts of proteolytic activ-
i 3 8
8,000
Y :
6,000
i
Substrate
TIME,
rut
blank
HOURS
FIG. 2. Relation bctwwn rnzymr~ amomlt and wtirity. Standard liver homogenate. (Amounts of tissue per ml homogenate.)
nssnq- proc~edur,~
:
220
ROTH,
LUSTY,
AND
-----------------
I
I I
U’IERBICKI
Substrate
/
1
I
2
3
4
TIME.
rat
FIG. 3. Kclation gkrocnrmus
ity are measured more.
between enzyme muscle homogenate.
the linearity
blank
amount, and (Amounts
HOURS
actirit,y. Standard of &sue per ml
of the reaction
assay procedure homogenate.)
may continue
;
for 24 hr or
DISCUSSION
The use of ‘“C-labeled hemoglobin as a substrate for determining total proteolytic activity offs 1rLv several advantages over present methods. The trchnique of labeling is simple and the substrate is stable and may be stored for long periods of time. Over t,no years’ experience in our laborat,ory wit’h this substrate embodying thousands of assays has demonstrated that the method is reproducible, quick, and rcliablc. In addition, we have used it to assay samples with prot’colytic activity so low that it is doubtful that it could have been detected by other mct~hotls. It is important to point out that this method, as others which use an added substrate, does not measure autolysis. It is clear from experiments carried out in our laboratory that proteolytic autolysis is inhibited by addition of an additional protein substrate, and what one is measuring is the ability of the tissues to degrade this added sub~trwtc to acid-soluble products. It is possible that the method may be applied to other special substrates, either natural or synthetic, with the same or other advantages. \T’e have tried, however, 14C-labeled lysozymc. which we found to be
The ineasurenient of total tiww proteolyt~ic :ivtivitp with :L hemoglobin substrate labeled with K1’CSO is dcwribcd. The labeled hemoglobin is stable and ik use offers sereral advanltnge~~ particularly in qwificGt,y and sensitivit,y owr incthods c*omi~onlp in me.
This work has been supported by the U. 8. Army Natick Laboratories undts1 contracts D$ IO-12%AMC-594 (S) anri I>&~(:-17-67-C-0156 with the Universit! of Connecticut. Jay Y. Roth was the l~rin(iI)al in\-estigator and Eugrn Wicrbicki, Chief, Irradiated Food Products Division, Food J&oratory, U. 8. Army Natirk Laboratories, was the Projwt OlIiwr for the subject research contracts. J. S. Roth is also a recipient of Curwr A\v:ud RC 63-13 from the Xationnl Instituiw of Health. S:ll ional Cancer Instit,ute.
1. 2. 3.
hl. I,.. J. (;etc. I’h&Yid. 22, 8 (l%s). H. I%, .4ND DIMLIS, J. T.. ,!&ci~r~. d. 87, 403 (1963). DE DUVE, C., PRESSMAN, B. C., GIAN~TO, R., Wa~~~arrx, R., AND AWLFZIINS, Uiochra. J. 60, 604 (1955). 4. PI,.\sT.~. R. J., .IND (;RUBICR, >I., BMLZ. Liiochen~. 5, 360 (1963). 5. %r.mli. G. I<.! STEIN, m-. H., AND RIOORE, S., J. Bid. (Ihem. 235, 3177 (1960). ;1NSON.
FELL,
I;.,