Darryel L. Reigh University of Colorado ~edicalschool Denver, 80220
I Brornolain
I
Experiments illustrating proteolytic enzyme action Experimental Possibilities
The following set of exercises provides a simple visual method of demonstrating some of the characteristics of enzymes in general, such as heat and p H lability and inhihition, as well as some specific properties of hromelain including some insight into the active site mechanism of peptide hydrolysis. These experiments have been utilized in both freshman general chemistry and junior level hiochemistry laboratories. Certain concepts presented should also he beneficial to high school students and college nonscience maiors.
Procedure or Treatment
elation
control Effect of untreated bromelain Heat stability
1mlHO I ml "'treated juice Heat 1 ml of juice for 5 mi". in a boiling water ban. Freeze 1 m l of juice i n a n ice salt mixture for
-
c o l d stability
&sic
Proteases are a group of enzymes capable of cleaving the peptide linkages of proteins. Gelatin, a protein prepared from collagen, is readily hydrolyzed by plant proteases ineluding bromelain, a proteolytic enzyme of members of the family Bromeliaceae. This ability of enzymes of certain plants to hydrolyze gelatin inspires the message "do not use fresh or frozen pineapple" on the back of each Jell@ package. This message could also apply to papaya as well as pineapple which contain the proteases papain and hromelain, respectively. An attempt to contradict the package warning leads to an understanding of some fundamental properties of enzymes. Experimental
Preoaration of Enzvme Source Fifty grams of fresh pineapple and 35 ml of sodium phosphate buffer (pH 7.0) containing 2 mglml of cysteine were homogenized in a blender for 1 min. The mixture was centrifuged at 8000 x G for 10 min and the supernatant liquid filtered through Whatman # 1filter paper to remove pulp. Effect on Gelatin The basic test calls for 1 ml of juice as prepared above and 1 ml of douhle-strengthjello (jello is dissolved according to package directions with one-half the normal amount of water and allowed to cool to approximately 50°C). To separate tubes add 1 ml of water and 1 rnl of . iuice.. then 1 ml of iello to each. Allow to stand for 5 min, then place in an ice bath. Gelation occurs rapidly in the control tube hut gelation proceeds very slowly and usually not at all in the tube containingjuice. Some experimental possibilities are summarized in the tahle. It is suggestedthat first all of the samples be prepared as listed, then followed by the addition of jello to each, and placed together in an ice bath. Time required for gelation and the degree of gelation is observed and recorded far each. 7
Discussion
In the experimental procedures descrihed in the tahle no gelation was observed for the tube containing untreated pineapple juice. The heated tube allowed gelation to occur and indicated that hromelain activity was destroyed on heating. This is a general property of most enzymes and exlai ins whv canned ~ i n e a ~would ~ l e be suitable for use in ;ello desserts since heattreatment is involved in the can-
a
386 / Journal of Chemical Education
+
+ -to
slight +
-to
slight +
-to
slight +
-1 -0 -in.. .....
pH st,abilitY
Introduction
~
concept
Acidic
Heavy metal inhibition S ~ l f h y d r y inhibil tion
Add 5 drops of 6 N NaOH t o 1 ml o f juice. Let stand 5 min. Add 5 a r m s of 6 N HCI. Add 5 drolm of 6 N HCI. Let stand 5 min. ~ d 5d FOPS 6 N NaOH. ~ d 5 adrops 2 % H9(N03)$. ~ d 0.1 a mgpchloramercuribenzoate.
+ +
ning process. Bromelain is only slightly affected by freezing which makes frozen pineapple unsuitable for desserts. Treatment with acid or base has only a slight effect suggesting a enzyme structure. The active site of hromelain contains a cysteine resiwith the sulfhydryl group postulated to ~ a r t i c i ~ a t e in peptide hydrolysis by the formation of a thio ester with the peptide bond. Heavy metal ions such as Hg2+, C U ~ + , and Ph2+ as well as compounds that form covalent complexes with free sulfhydryl groups such as p-chloromercurihenzOate4 have heen shown to inhibit hromelain action and of hromethese observations are confirmed by the lain treated with Hg(NO& and p-chloromercurihenzoate to prevent gelation. Further Studies
Other proteolytic enzyme actions can he demonstrated using the procedure proposed above. Adolph's@meat tenderizer which contains papain and Axion@detergent presoak which contains a mixture of proteolytic enzymes can be substituted for pineapple juice in these experiments. An expanded heat stability study of hromelain or papain which are both available commercially from Sigma Chemical Company could also he devised.
' Hollaway, M.92,R., Mathias, A. P., and Rabin, B. R., Bioehern. (1964),
Biophys,
Wang, R. C., and Leiner, I. E., Bioehem. Biophys. Res. Comm., 17,470 (1964). Stern. K. G.. Biachern. Z.. 234.116 (19311. ~eini'cke, R.'M., and oh, ~ . , . ~ e i e n ei29,1678 e, (1959).
'
I Brornolain
I
Experiments illustrating proteolytic enzyme action Experimental Possibilities
The following set of exercises provides a simple visual method of demonstrating some of the characteristics of enzymes in general, such as heat and p H lability and inhihition, as well as some specific properties of hromelain including some insight into the active site mechanism of peptide hydrolysis. These experiments have been utilized in both freshman general chemistry and junior level hiochemistry laboratories. Certain concepts presented should also he beneficial to high school students and college nonscience maiors.
Procedure or Treatment
elation
control Effect of untreated bromelain Heat stability
1mlHO I ml "'treated juice Heat 1 ml of juice for 5 mi". in a boiling water ban. Freeze 1 m l of juice i n a n ice salt mixture for
-
c o l d stability
&sic
Proteases are a group of enzymes capable of cleaving the peptide linkages of proteins. Gelatin, a protein prepared from collagen, is readily hydrolyzed by plant proteases ineluding bromelain, a proteolytic enzyme of members of the family Bromeliaceae. This ability of enzymes of certain plants to hydrolyze gelatin inspires the message "do not use fresh or frozen pineapple" on the back of each Jell@ package. This message could also apply to papaya as well as pineapple which contain the proteases papain and hromelain, respectively. An attempt to contradict the package warning leads to an understanding of some fundamental properties of enzymes. Experimental
Preoaration of Enzvme Source Fifty grams of fresh pineapple and 35 ml of sodium phosphate buffer (pH 7.0) containing 2 mglml of cysteine were homogenized in a blender for 1 min. The mixture was centrifuged at 8000 x G for 10 min and the supernatant liquid filtered through Whatman # 1filter paper to remove pulp. Effect on Gelatin The basic test calls for 1 ml of juice as prepared above and 1 ml of douhle-strengthjello (jello is dissolved according to package directions with one-half the normal amount of water and allowed to cool to approximately 50°C). To separate tubes add 1 ml of water and 1 rnl of . iuice.. then 1 ml of iello to each. Allow to stand for 5 min, then place in an ice bath. Gelation occurs rapidly in the control tube hut gelation proceeds very slowly and usually not at all in the tube containingjuice. Some experimental possibilities are summarized in the tahle. It is suggestedthat first all of the samples be prepared as listed, then followed by the addition of jello to each, and placed together in an ice bath. Time required for gelation and the degree of gelation is observed and recorded far each. 7
Discussion
In the experimental procedures descrihed in the tahle no gelation was observed for the tube containing untreated pineapple juice. The heated tube allowed gelation to occur and indicated that hromelain activity was destroyed on heating. This is a general property of most enzymes and exlai ins whv canned ~ i n e a ~would ~ l e be suitable for use in ;ello desserts since heattreatment is involved in the can-
a
386 / Journal of Chemical Education
+
+ -to
slight +
-to
slight +
-to
slight +
-1 -0 -in.. .....
pH st,abilitY
Introduction
~
concept
Acidic
Heavy metal inhibition S ~ l f h y d r y inhibil tion
Add 5 drops of 6 N NaOH t o 1 ml o f juice. Let stand 5 min. Add 5 a r m s of 6 N HCI. Add 5 drolm of 6 N HCI. Let stand 5 min. ~ d 5d FOPS 6 N NaOH. ~ d 5 adrops 2 % H9(N03)$. ~ d 0.1 a mgpchloramercuribenzoate.
+ +
ning process. Bromelain is only slightly affected by freezing which makes frozen pineapple unsuitable for desserts. Treatment with acid or base has only a slight effect suggesting a enzyme structure. The active site of hromelain contains a cysteine resiwith the sulfhydryl group postulated to ~ a r t i c i ~ a t e in peptide hydrolysis by the formation of a thio ester with the peptide bond. Heavy metal ions such as Hg2+, C U ~ + , and Ph2+ as well as compounds that form covalent complexes with free sulfhydryl groups such as p-chloromercurihenzOate4 have heen shown to inhibit hromelain action and of hromethese observations are confirmed by the lain treated with Hg(NO& and p-chloromercurihenzoate to prevent gelation. Further Studies
Other proteolytic enzyme actions can he demonstrated using the procedure proposed above. Adolph's@meat tenderizer which contains papain and Axion@detergent presoak which contains a mixture of proteolytic enzymes can be substituted for pineapple juice in these experiments. An expanded heat stability study of hromelain or papain which are both available commercially from Sigma Chemical Company could also he devised.
' Hollaway, M.92,R., Mathias, A. P., and Rabin, B. R., Bioehern. (1964),
Biophys,
Wang, R. C., and Leiner, I. E., Bioehem. Biophys. Res. Comm., 17,470 (1964). Stern. K. G.. Biachern. Z.. 234.116 (19311. ~eini'cke, R.'M., and oh, ~ . , . ~ e i e n ei29,1678 e, (1959).
'
