INSTITUTO POLITÉCNICO

NACIONAL
ESCUELA NACIONAL DE
CIENCIAS BIOLÓGICAS

LABORATORIO DE MÉTODOS DE
ANÁLISIS

PRÁCTICA:
“DETERMINACIÓN DE LA ACTIVIAD DE
ENZIMS PROTEOLÍTICAS. MÉTODO DE
KUNITZ”

Alum!: Am"#! B$"%#&' U#&$l

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FUNDAMENTO DEL MÉTODO DE KUNITZ
El m=%!7! /$ u7"m$%" $ $l ,"m3&! 7$ /!lu3&l&7"7 7$ u" (#!%$>" $ ?,&7!
8
%#&,l!#!",=%&,! @ATC ,u"7! $/%? /u$%" " l" ",,&- 7$ u" $'&m" (#!%$!l&%&,".
M&7$ l" ,"%&7"7 7$ (=(%&7!/ 9! "m&!?,&7!/ 0u$ /$" /!lu3l$/ 7u#"%$ l"
6&7#-l&/&/. D&,6!/ ,!m(u$/%!/ ! "l m$!/ "l4u!/ "3/!#3$#? " 8 m.
OBJETIVOS
• E/%"3l$,$# u" ,u#1" 7$ ",%&1&7"7 $'&m?%&," $/%?7"#.
• D$%$#m&"# l" ",%&1&7"7 $'&m?%&," 7$ l" 3#!m$l">"  l" ("(">" @$'&m"/
(#!%$!l>%&,"/ " ("#%&# 7$ u" ,u#1" $/%?7"# 7$ ",%&1&7"7 $'&m?%&," 7$ l"
T#&(/&".
• D$%$#m&"# ,u?l 7$ l"/ $'&m"/ $m(l$"7"/ %&$$ m"!# ",%&1&7"7
$'&m?%&," /!3#$ $l m&/m! /u/%#"%!.
RESULTADOS
Tabla 1. D"%!/ ("#" l" ,u#1" 7$ A,%&1&7"7 $'&m?%&," 7$ l" T#&(/&"  7$ l!/ P#!3l$m"/ 7$
l"/ $'&m"/ " 7$%$#m&"# /u ",%&1&7"7.
Conc. de A280 A280
Tubo Tripsina Problem Testigo corregid
(mg/mL) a a
T1 y 1 0.04 0.221 0.049 0.172
T2 y 2 0.08 0.367 0.051 0.316
T y  0.12 0.455 0.059 0.396
T! y ! 0.16 0.549 0.054 0.495
T" y " 0.2 0.588 0.067 0.521
Problemas
T# y # $romela%na 0.727 0.44 0.287
1&10
T## y ## $romela%na
0.417 0.269 0.148
1&20
Tlll y lll Papa%na 1&2 1.147 0.331 0.818
Tl' y #' Papa%na 1&! 0.681 0.216 0.465
 12

10

A280 corregida 6

0
0.020 .040.0
() 60.
* 08 0.1 0.120. 140. 160. 18 0.2 0.22
+, * 0
Concentraci-n de T
ripsina (mg/mL)

Gráfica 1. Cu#1" 7$ A,%&1&7"7 $'&m?%&," 7$ l" T#&(/&". S$ %!m"#! $ ,u$%"

2&,"m$%$ l!/ 7"%!/ 7$ "3/!#3",&" ,!##$4&7" 7$ l!/ %u3!/ T+  + "l TF  F. @ Ver
discusión.

Ui!a!"# Tr$%&ica#' P$7&$%$ 7$ l" #$,%"  8.<88)  (.1)11 UT*+,

8 m&. 8 m&.

Tabla -. D"%!/ ("#" l" Cu#1" 7$ ",%&1&7"7 $'&m?%&," $/($,>&," ("#" l" 7&4$/%&- 7$
,"/$>" (!# %#&(/&".
 A280
nidades
Tubo corregid
Tr%pticas.
a
1 0.005245 0.172
2 0.01049 0.316
 0.015735 0.396
! 0.02098 0.495

0.

0."
() * 20  0.08
+, * 0.8
0.!

A280 corregida 0.

0.2

0.1

0
0 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02

nidades Tripticas
 Gráfica -. Cu#1" 7$ ",%&1&7"7 $'&m?%&," $/($,>&,". S$ %!m"#! $ ,u$%"
2&,"m$%$ l!/ 7"%!/ 7$ l!/ %u3!/ T+  + "l TF  F. @ Ver discusión.
Tabla ). UT94 7$ mu$/%#" $ (&H"  ("("" @3#!m$l">"  ("(">" #$/($,%&1"m$%$
T T/gde
muestra&
$romela%na
0.010225 0.12679
1&10
$romela%na
0.003275 0.08122
1&20
Papa%na 1&2 0.036775 0.064724
Papa%na 1&! 0.019125 0.06732

álc/l0#'

A3/!#3",&" ,!##$4&7": A280 problema - A280 testigo 0.221 – 0.049 = 0.172

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D$/(u=/ ("#" l!/ UT94 mu$/%#":

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El u/! 7$ l!/ %u3!/ %$/%&4!/ $/ (#&,&("lm$%$ $l 7$ %$$# %u3!/ /& ",%&1&7"7
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7$ "3/!#3",&" 7$3&7! " 0u$ 7$/(u=/ 7$ #$"l&'"# l" 6&7#-l&/&/ $ l!/ %u3!/
(#!3l$m" l" /!lu,&- "3/!#3$#? ,!m! /& =/%" %u1&$#" u" ,"%&7"7 7$ (#!%$>"
m"!# " l" 0u$ (u/&m!/. P!# l! %"%! 7$3$ !3%$$#/$ u" "3/!#3",&" ,!##$4&7"
/&$7! $/%" l" 7&$#$,&" $%#$ l" "3/!#3",&" 7$l (#!3l$m"  l" "3/!#3",&" 7$l
%$/%&4! l! "%$#&!# /$ 6",$ ,! l" &"l&7"7 7$ !3%$$# #$/ul%"7!/ $J($#&m$%"l$/
m?/ ,!&"3l$/.
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/$ (u$7$ u%&l&'"# ("#" ,!!,$# l" ",%&1&7"7 (#!%$!l>%&," 7$ !%#"/ $'&m"/ ,!m! l"
("(">"  3#!m$l">" (u$/%! 0u$ l!/ 7"%!/ 7$ 7&,6" ,u#1" /! &7$($7&$%$/ 7$
l" $'&m" (#!%$!l>% ," u%&l&'"7" /!3#$ $l /u/%#"%! @$ $/%$ ,"/! ,"/$>" /&$m(#$ 
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 )C.

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$/%u7&"m!/ !3/$#1"m!/ 0u$ =/%"/ %&$$ u" m"!# ",%&1&7"7 (#!%$!l>%&,"
@6&7#!l&'" m?/ " l" ,"/$>" (u$/%! 0u$ $l 1"l!# 7$ UT ,"l,ul"7!/ /! m"!#$/ "
l!/ !3%$&7!/ ("#" l" %#&(/&". Au0u$ 6" 0u$ ,!/&7$#"# 0u$ l!/ 1"l!#$/ 7$ UT
("#" l" ("(">"  3#!m$l">" l"/ !3%u1&m!/ (!# 4#"m! 7$ mu$/%#". O3/$#1"m!/
"7$m?/ 0u$ (!# %$$# u" m"!# ",%&1&7"7 (#!%$!l>%&," l"/ $'&m"/ %#"3""7"/ l"/
,!,$%#",&!$/ 7$ UT ! $%#" 7$%#! 7$ l" ,u#1" 7$ ",%&1&7"7 $/($,>&,".
C!m("#"7! l" ",%&1&7"7 $'&m?%&," 7$ l" ("(">"  l" 3#!m$l">" !3%u1&m!/ 0u$
=/%" 2l%&m" %&$$ m"!# ",%&1&7"7 $'&m?%&," (u$/%! 0u$ %&$$ 1"l!#$/ m?/ "l%!/ 7$
UT94 7$ mu$/%#". L! "%$#&!# (!7#>" #$l",&!"#/$ ,! 0u$ =/%"/ 8 $'& m"/ /!
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$'&m?%&," /&m&l"# " l" 7$l %u3! m$!/ 7&lu&7! @+:8.
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6&7#!l>%&,"/ 0u$ =/%$ ,!%&$$. L" (&H" 7$ l" ,u"l 6&'! $l $J%#",%! $/%"3" mu
m"7u#"
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7$ l" $J(l&,"# %"m3&= (!# 0u= $/ m"!# l"
3#!m$l">".
ONLUSIONES
S$ (u$7$ !3%$$# u" ,u#1" 7$ ",%&1&7"7 $'&m?%&," $/%?7"# 0u$ /$"
&7$($7&$%$ 7$ l" $'&m" (#!%$!l>%&," /&$m(#$  ,u"7! /$ u%&l&,$ $l m&/m!
/u/%#"%!  l"/ m&/m"/ ,!7&,&!$/ $J($#&m$%"l$/ @( T$m($#"u#" $%,.
S$ !3%u1! u" ",%&1&7"7 $'&m?%&," ("#" %#&(/&" 7$: .+++ UT9m4
S$ !3%u1! u" ",%&1&7"7 $'&m?%&," ("#" 3#!m$l">" 7$: .+8<  .+ UT94 m%".
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5REGUNTAS ADIIONALES
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7 Cu?l $/ l" $/($,&&,&7"7 7$ $l",$ 7$ l" %#&(/&"
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BIBLIOGRA5ÍA:
+. S%$# L. B&!0u>m&,". < $7&,&-. R$1$#%=. B"#,$l!". 8. P(. <Q.
8. Ramírez !. et al. "#$%&A'%(A ) *" A('%+%*A*") ,R'"%'%(A) *"
,R*/(') /'%%$A#* A'" *" ,A,AA (aria ,apaa ,ARA RAR "
AA#*A&%"#' *" A (AR#". " líea *epartameto e ra:aos e
%;estigai< e Alimetos. "#(-%,#. (os:lta 18>09>2016. *ispoible e
?@ttp>>.smbb.om.mB>ogresos
C20smbb>morelia07>'RAAD)>AreaE%%%>(arteles>(%%%-8.pF G
3. (la;iHo *iegoI ,ortilla &artiez &ag@iel (eiliaI J:iHao ,arra AlFoso (iKtia
e la bromelia obteia a partir e la piLa perolera Aaas (omos:s e
ebriHa-)ataer ist:a Re;ista e la Ma:lta e (ieias Nsias ;ol. 10
Om. 2 2012 pp. 41-49