UNIVERSIDAD DE CHILE FACULTAD DE CIENCIAS DEPARTAMENTO DE QUÍMICA BIOQUÍMICA Y QCA. BIOLÓGICA
Cinética enzimátic a Determinación Determinación de la actividad enzimática y de la actividad específca de la osatasa acida del germen de trigo
Integrante!
S"#$a A%&'a(a V. )"* M+g&e, Le-n
Fe%a (e entrega! /0 (e A1r+, (e /234
Introducción La #"#ataa5 "n 6arte (e &n gr&6" (e en7+'a ,,a'a(" eteraa5 ,a 8&e a & 9e7 "n &na &1ateg"r+a (e ,a %+(r",aa. La %+(r",aa5 "n a8&e,,a en7+'a 8&e ata,+7an &na rea+-n (e %+(r",++. En tant"5 ,a #"#ataa a, er 6arte +ntegrante (e e,,a5 ata,+7an &na rea+-n en ,a &a, e ,+1era &n gr&6" #"#at" (e, &trat" : e (e;a a ete "n &n gr&6" %+(r"<+," 'e(+ante ,a rea+-n genera, '"tra(a a "nt+n&a+-n!
Figura 1. E&a+-n re6reentat+9a (e &na rea+-n ata,+7a(a 6"r &na #"#ataa
En e, +g&+ente tra1a;" 6r=t+" &t+,+7are'" ,a #"#ataa =+(a (e, ger'en (e tr+g"5 ,a &a, %+(r",+7a #"#"'"n"*tere ,+1eran(" "rt"#"#at" +n"rg=n+". Para eta en7+'a5 (a(a & 1a;a a>n+(a( 6"r ,a 6"r+-n "rg=n+a (e, &trat" e 6&e(en &t+,+7ar &trat" art+>+a,e5 en ete a"5 e, 6?n+tr"#en+,#"tat"5 e, &a, a, %+(r",+7are (ar= 6?n+tr"#en",. E, 6? n+tr"#en", en &n 'e(+" ," &>+ente'ente 1=+" @6H .34 5 ,+1era e, 6r"t-n (e, %+(r"<+," : 8&e(a e, +"n #en",at"5 e, &a, e ","rea(" : 6"r tant" a1"r1e en e, 9++1,e. Et" ,t+'" 6er'+t+r= eg&+r ,a at+9+(a( (e ,a rea+-n a tra9* (e &n e6etr"#"t-'etr" a 2 n' @,"ng+t&( (e "n(a arater$t+a (e, 6?n+tr"#en",. La rea+-n 8&e (er+1e ," ea,a(" anter+"r'ente!
Figura 2. H+(r-,++ 6?n+tr"#en+,#"#at" ata,+7a(a 6"r #"#ataa. F"r'a+-n 6?n+tr"#en", en &
#"r'a ","rea(a
F+na,'ente5 ," "1;et+9" (e, +g&+ente tra1a;" e<6er+'enta, "n (eter'+nar e, "e>+ente (e ea (e ,a ",&+-n (e #"#ataa =+(a5 6ara et"5 (e>n+re'" &na &n+(a( (e at+9+(a( en7+'=t+a "n+(eran(" ta'1+*n ," "ntr",e a rea,+7ar @1,an" (e &trat"5 "ntr", n" en7+'=t+" : t+e'6" er".
Resultados y análisis de resultados En ,a ta1,a a(;&nta5 e 6reenta ,a +n#"r'a+-n aera (e ,a ant+(a( (e 6?n+tr"#en", 42 M5 a(e'= (e ,a ant+(a( (e NaOH 253 M 8&e #&e agrega(a a a(a t&1". Ta'1+*n e (eta,,a & "nentra+-n : a1"r1an+a a 2 n'. Tabla 1. A1"r1an+a 6ara (+#erente "nentra+"ne (e 6?n+tr"#en",
# Tub o
Cantidad de pnitrofenol 6 !" agregada $%&
Cantidad de 'a() *1 " agregada $%&
Concentración de p-nitrofenol n$ol+6$%&
,bsorbanci a a n$
3 / J
3 / J
J / 3
42 3/2 302 /2 J22
253JK 25/ 25/ 25 25/3
E'6,ean(" ,a "nentra+-n (e 6?n+tr"#en", : ,a a1"r1an+a "rre6"n(+ente5 e 6r"e(e a e,a1"rar ,a &r9a (e a,+1ra+-n. A(e'=5 e "n+(era e, a8&e, 6&nt" en ("n(e ,a "nentra+-n (e 6?n+tr"#en", e 2 8&e "rre6"n(e a &na a1"r1an+a (e 2.
Figura 2. C&r9a (e Ca,+1ra+-n 6ara e, 6?n+tr"#en",.
La 6en(+ente (e ,a reta "rre6"n(e a, 9a,"r (e, "e>+ente (e en (e 8&e ,a en7+'a ata,+e ,a rea+-n (e %+(r-,++ (e, 6?n+tr"#en+,#"#at". Para e,," e +ntr"(&en (" t&1" (e ena:" (eter'+na(" 9",'ene (e +trat" (e "(+"5 ag&a5 6?n+tr"#en+,#"#at" : #"#ataa a,a,+na (e ger'en (e tr+g" %ata "'6,etar &n 9",&'en (e ",&+-n (e 25'L. La +n&1a+-n e ,,e9" a a1" 'e7,an(" en 6r+'er ,&gar e, 9",&'en (e +trat" (e "(+"5 6?n+tr"#en+,#"#at" : e, ag&a en &n t&1" (e ena:" A5 e, &a, e +ntr"(&e en e, 1a" (e ag&a a,+ente a J
℃
6"r (" '+n&t". A, tran&rr+r
," (" '+n&t" e +ntr"(&e r=6+(a'ente ,a en7+'a5 : e +n&1a 6"r '+n&t" a J
℃
. A, ter'+nar ," '+n&t" e agregan 5'L (e NaOH 253M e, t&1" A
6ara (etener ,a rea+-n. Se re6+te ete 6r"e(+'+ent" "n e, t&1" (e ena:" B5 6er" "n ,a (+#eren+a 8&e e +n&1a "n ,a en7+'a 6"r 32 '+n&t". Tabla 2. Volumen de cada componente utilizado para la incubación.
T&1"
V",&'en 'L
Tie$po
En7+'a
C+trat" (e Na
"inutos 6? H/O n+tr"#en+,#"#at " 25/2 'g'L 253M 6H /5'M A 253 25/ 253 253 B 253 25/ 253 253 1 Se agrega a a(a t&1" &n 9",&'en (e 5'L (e NaOH a, ter'+nar e, t+e'6" (e +n&1a+-n >;a(" 6ara a(a t&1".
Se "1er9a &na ",&+-n tran6arente : (e ","r a'ar+,," ,ar" en e, t&1" A5 e, &a, #&e +n&1a(" 6"r '+n&t" en e, 1a" a J
℃
. En a'1+" e, t&1" B
+n&1a(" 6"r 32 '+n&t"5 6reenta &n ","r a'ar+,," &n 6"" '= +nten". Se 6r"e(e a rea,+7ar J t&1" "ntr",e5 6ara e,areer + (entr" (e ,a ",&+-n 6re6ara(a e<+te &n "'6"nente (+#erente a ,a en7+'a 8&e 6&e(a ,,e9ar a a1" ,a rea+-n. L" "ntr",e rea,+7a(" "n+ten en5 &n "ntr", n" en7+'=t+" @t&1" C5 en e, 8&e e &t+,+7a ,a 'e7,a (e rea+-n en ,a &a, ,a en7+'a e ree'6,a7a(a 6"r ",9ente. Otr" "ntr", e e, (e, t+e'6" er" @t&1" E5 en e, 8&e ,a rea+-n e (et+ene en e, '+'" '"'ent" (e "'6,etar,a5 agregan(" e, +n%+1+("r ante (e ,a en7+'a. Y 6"r ,t+'" e, 1,an" (e &trat" @D5 en ete "ntr", e ree'6,a7a e, &trat" 6"r e, ",9ente. L" t&1" C : D e +n&1an en e, 1a" a,+ente a J
℃
5 e, t+e'6" (e
+n&1a+-n e (+#erente 6ara a(a &n"5 e, t&1" C e (e;a &n ,arg" t+e'6" 3 '+n&t"5 : e, t&1" D e (e;a en e, 1a" a,+ente 6"r '+n&t". E, t&1" E n" e +n&1a.
Tabla 3. Volmenes utilizados en la realización de controles enzimáticos
T&1" En7+'a C D E
25/2 'g'L ? 253 253
V",&'en 'L C+trat" (e Na 6? n+tr"#en+,#"#at" 253M 6H /5'M 25/ 253 ? 253 25/ 253
H/O 25/ 25/ 253
L&eg" (e rea,+7ar t"(" ," ena:" en7+'=t+" : ," "ntr",e re6et+9"5 e '+(e ,a a1"r1an+a (e a(a &na (e ,a ",&+"ne "1ten+(a a 2n'. Tabla !" #bsorbancia medida para cada uno de los tubos
T&1" A1"r1an+a n' A2
A 253/
B 2530
C 252/0
D 252/2
E 252/
De ," 9a,"re "1ten+(" 6"(e'" (arn" &enta 8&e n" %a: at+9+(a( en7+'a5 a, e,+'+nar ,a en7+'a (e ,a ",&+-n5 : ta'1+*n a, e,+'+nar e, &trat". Et" 8&+ere (e+r 8&e n" %a: "'6"nente en ",&+-n 8&e 6&e(an a,terar ,a at+9+(a( ata,$t+a (e ,a en7+'a (e 'anera 6"+t+9a " negat+9a. Et" ," "'6r"1a'" "n e, "ntr", a t+e'6" er"5 ("n(e en ",&+-n e en&entra e, &trat" : ,a en7+'a5 6er" a (+#eren+a (e ," t&1" &t+,+7a(" en e, ena:" en7+'=t+" a ,a 'e7,a e ,e agreg- 6re9+a'ente NaOH5 "'6"nente 8&e &'6,e "n ,a #&n+-n (e (etener ,a rea+-n5 et" e rea,+7- "n ,a +nten+-n (e (etetar a,g&na +nter#eren+a 6r"9"a(a 6"r &a,8&+er "'6"nente (e ,a rea+-n5 8&e n" ea e, &trat" : ,a en7+'a5 :a 8&e et" et=n (earta(" 6"r ," "ntr",e %e%" ante. Se "n,&:e 8&e en ,a ",&+-n n" %a: "'6"nente 8&e 6&e(an a,terar ,a at+9+(a( ata,$t+a (e ,a en7+'a #"#ataa. E (e e6erar 8&e ,a a1"r1an+a 'e(+(a a ," "ntr",e C5 D : E ea +(ea,'ente er"5 6er" n" "&rre5 :a 8&e en e, &1" 6&e(e etar 'a, ,a9a(" : 6"r e,," "ntener a,gn "'6"nente5 e, &a, e a6ta(" 6"r e, e6etr" (e a1"r1an+a5 e 6"r et" 8&e ," 9a,"re et=n &n 6"" '= a,t" 8&e er". En e, a" (e, t&1" A : B5 a, 'e(+r ,a a1"r1an+a (e, t&1" A a ," '+n&t" : ,&eg" 'e(+r a (e, t&1" B :a tran&rr+(" 32 '+n&t" (e rea+-n5 e ree 8&e ,a a1"r1an+a (e1er$a (ar e, ("1,e5 :a 8&e tran&rr+(" e, ("1,e (e t+e'6"5 e (e1er$a #"r'ar e, ("1,e (e 6r"(&t". Et" n" "&rre : (a'" ra7-n a et" a, err"r %&'an" 8&e 6&e(e e<+t+r a, '"'ent" (e e'6e7ar a 'e(+r e, t+e'6"5 " a, agregar ,a en7+'a. A tra9* (e ,a &r9a (e a,+1ra+-n rea(a5 e a,&,a ,a "nentra+-n (e 6? n+tr"#en", #"r'a("5 +nter6",an(" ,a a1"r1an+a 'e(+(a en 6ara a(a t&1".
Tabla $" #bsorbancia y concentración del control enzimático.
T&1"
A2 n'
A B C D E
253/ 2530 252 252 252
C"nentra+-n (e 6? n+tr"#en", n'",e4'L / 2 2 2
