。風
0.61
。31 ωQSAEZ〈 。
0、
270 且，90 310
Wavelength (nm) •
Fi郎ue2: The absorption band ofTRITON X-I00.
ZQ ~30
0
•
九
。、
。、
。OSRHHSA〈
0.'
Figure 3: The UV - Record of Sheep Seium Albumin.
3)0
2.10 2!JQ 310
Wavelength (nm)
。 2防
。
( 319 )
2. Preparation of TRITON X-l 00 solutions:
200 戶cc of TRITON X-I00 were added to 200 ml buffer solution and di1uted to desired concentrations. The CAR Y 118 spectrophotometer was used to measure its absorbance from which the concentration can be calculated. The
師一-一，
大學 報 第
absorption band ofTRITON X-I00 is shown in figure 2.
3. Preparation of membranes:
Membranes were prepared by the method of McPhie19 to remOve
士 soluble UV absorbing substances. The process is as follows:
期 (a) Boi1 membranes in 50% H20 - C2HsOH solution gently about .1 hour, with
sti汀ing.
(b) Drain out the solution and rinse the membranes 3 times with glass disti11ed wàter.
(c) Repeat (a) (d) Repeat (b)
(e) Boil membranes in 10 mM NaHC03 solution about 1 hour, with stirring.
(f) Repeat (b) (g) Repeat (e) (h) Repeat (b)
(i) Boi1 membranes 姐 1 mM EDTA solution about 1 hour, with sitr也可
(j) Repeat (b)
(k) Boi1 membranes in glass distilled water about 1 hour, with stirring.,
(1) Repeat (b) (m) Repeat (k) (n) Repeat (b)
4. Preparation of protein solution:
Dissolve 0.7 mg of N-Ethylmaleimide (NEM) in 1 ml of pH 7.0
phos-尺 phate buffer solution. Add 0.17 gm of p抖pro削.0枷叫 7m叫1 addi址iti“iona叫1 bu吋f叮f叮叭叩叫olu吋I泊t位i tωo dissolve albumin. Stir regularly every 10 minutes or less. Reaction is continued for 1.5 hours at room temperature. This blocks the sulfuydryl on the albumin.
The solution of mixed protein and NEM is transferred to a dialysis bag and placed in the buffer solution with gentle stirring at 40C to remove excess NEM. Removal
( 320 )
V
limit [A]
(12)
=
U1,吋HNH吋OZMtso典人血清蛋白素及羊的抖清蛋白索恆溫下結合之研究
一一二-2U~ U1
U
1[A] • 0
d(一~.
-')limit '[A]' = [A]•
o
d V(13)
Equations (12) and (13) tell us that the Scatchard plot can be used to estimate the frrst two association constants for the very low concentration of the low molecular weight molecule.
MATERIALS AND METHODS
Materials
The TRITON X-I00 wa.s obtained from Sigma Chemical Corporation (Lot Ill.
、••
J
•••
A
rs.、
#95 L-0059) and the HSA was purchased from Miles Laboratory (Fraction V, Lot #136)
,
SSA was a1so obtained from Miles Laboratory (Fraction V,
Lot #13).These two proteins were used without further purification. In dialysis cells semi-permeable membranes were used to separate the protein solution, TRITON X-l 00 solution and buffer solution. The membranes were bought from Union Corpora-tion, Chicago, Illinois. (#3787-042, 12,000 molecular weight cut-off). All water used was disti11ed twice from deionized. water. The equilibrium dialysÎS was carried out in pH 7.0 phosphate buffer. All ultra-violet (UV) absorbance measurements were made wiht a CARY 118, UV spectrophotometer.
Experimental Methods (2)
Preparation of fresh phosphate buffer solution:
Stock solutions of 0.5M of KH2P04 (68.05gmjl000cc) and 0.5M of
七
Na2HP04 (70.98 gmjl000 cc) were prepared and stored at 40C. Phosphate buffer was prepared by taking 22.4 cc of the above KH2 P04 solution mixed with 25..8 cc of Na2HP04 solution di1uted to 1 liter. This last solution has a pH value equal to 7.0. 四lepH was routinely checked with a Beckman 3500 pH meter.( 321 )
slope n ~ 1, the sites of the macromolecule are independent, and if slope n
>
1the binding is highly cooperative
,
Suppose the macromolecule has m c1asses of binding sites and Ni. is the number of binding sites in c1ass i. According to the Scatchard model, one obtains
Ki
[A]
(9) 1 + Ki [ A]from the formulation.
m
F =z
Ni師大學報
where Ki is the average association constant for that class. When applying the
,Bcatèhard model to the binding of largeorganic ligands on proteins, the assumption made is that all of the binding sites exist initially and are completely independent.
This is inconsistent with the data that the binding of certain organic ligands to
第二十三期
albumin produces conformational change in which binding sites are altered or . Therefore, any information we obtain must be derived from the Adair equation which is the general case and not constrained by any assumption.
From equation (5), v formed 1 6 -1 7
can be expanded in powers of [A] by binomial theory
I+U1 [A] 十 U2[A]2+...
as done in appendix 2, to give a polynomial.
U1 [A] + 2U2 [A]2 +. . . . + nUn[A]n + Un [A]n
v
=
-1
. = UdA] + (2U2-U12)[A]2 + (3U3-3U2U1+U13)[AP +(4U4-4UIU3-2U~2
+4U12U2 -U14)[A]4 +(5Us-5U4Ul-5UgU2+5UIU22 +5U12U3 -5U13
(10) whereK1 =U1,K1K2=U2,.. .. . .K1K2. .." Kn=Un
Let us write,
v
= a1 [A] + a2 [A] 2 + . . . +an [A] n-The coefficients ai's, can be obtained by a least square fitting. Then U 1, U 2,.., , Un can be calculated from al , a2, . . . ., ~ and consequent1y K 1 ,K2 ,.... ,Kn'
In another method used by Bartholmes et. al.悶，K 1 and k 2 can be evaluated U2 + U1 5) [A] 5 + . ... . .
-'-/、
from the Scatchard plot. From Appendix 2, we have
( 322 ) have the same value K. As derived in appendix 1,
吋HNH吋OZMSHOO典人血清蛋白素及羊血清蛋白素恆溫下結合之研究 Ki
=
(n 一i+1) K / i .and the average molar ratio can be simplified to equation (6)
(6a)
= Kn[A] / (1 + K[A]) v
[A] (6b)
v
and [A] can be measured, and plotting 二:-;versus一-V [A]a graph called a Scatchard plot6• If the Scatchard plot is linear, then it .implies yields
=
(n - v ) KIn equation (6b),
that the macromolecule has only independent and identical
association constant, whlch is a measure of binding strength between macromole-cule and small molemacromole-cule, is the slope of the Scatchard plot. The total number of
binding sites. The
binding sites on macromolecule, n, is evaluated by the extrapolation of the linear plot of equation (6b) to intercepts on either the abscissa or ordinate.
In order to verify that an experimental system is adequately described by the Scatchard model, the experimental data must span the range from 0..;;; v ~n.
This extrapolation is potential1y erroneous if limited data is used to defme the Scatchard plot1 5 .
Macromolecules may have cooperative and dependent sites. Jn other words, there are the interactions of one ligand with another where the binding at any site affects the binding affinity at other sites. First1y, we consider the case that the binding of a ligand on one site of a macromolecule activates so strongly .such that, the other sites will be filled up immediately. Therefore, only molecules P and PAn exist. Equation (5) becomes
= nK[A]n/ (1+K[A]n) v
(7) 五
) = K[A]n ln(一主士)
=
n ln [A] + ln Kn- v
v. /
(n-v
or
(8)
Plotting 扭(之一=)
versus ln[訓，
the resulting graph is called a Hill plot. If then-V
( 323 )
ratio of bound 1igand to the macromolecule and is defmed by the equation total (3)
= [A] bound/[P]
v
It is obvious rhat [A] bound and [P] total can be expressed as following.
師大學報
[PA] + 2 [PA2 ] + . . . + n [P~]
[A]bound =
(4) [PAn]
+ [PA]
[P] + [P] total 一
第二十三期
From equation (2), (3), and (4), wehave
1 +K1 [A] + K1 K2 [A] 2 +. . . + K1 K2 . • • • Kn[A] n n
~ J ,;
j=lj(ifl Ki)[A]J
K1 [A] + 2K1 K2 [A]2 +. . . + nK1 K2 • • • Kn[A] n v
-1
+豆 (L
L)[Alj (5) j= 1 'i= 1 1equation (5) is called Adair equation14
Let us first, consider the special case where all n sites of the macromolecule are identica1 and completely independent. For this reaction, P + A 三 PA， there would then be C? different forms of PA combination. Figure 1 is a brief explana.
tion of this situation.
i::|
、..
J
it Ku
|::l
(a)
Figure 1: 4-site macromolecule P has 4 different binding forms.
四
(a) n = 4, a11 sites are empty (b) 4 forms of 1 site fi1led.
In general, there
are 們 different
forms of the species P~﹒ Because
each site is identica1, we can assume that the association constant for identical sites would( 324) Albumin complexes will begin to answer this question.
吋同阿吋OZMntHg
與人血清蛋白索及羊血清蛋白索恆溫下結合之研究
The specific objectives of this investigatidn are:
for TRITON X-I00 binding to HSA and sheep serum albumin (SSA), at different concentrations ofTRITON X-I00.
To fit the binding isotherms to an appropriate binding model, (Scatchard To determine, v
2.
or a general stepwise equilibrium model).
To compare the binding mechanism for the two serum albumins with 3.
ofBSA.
that
n.
THEORYConsider a macromolecule protein P combining reversibly with a small weight molecule, A. The number of combining sites on the macromolecule may be large.
In general, it can be described by stepwise equilibrium reactions1 3 :
A~PA
P +
A~
PA2+ PA
、‘EJ
...
•. /
、A~ PA3
+ PA2
A~ PAn PAn-l+
are n association constants which reactions, there
For these equilibrium
describe the binding of the small molecule A to the macromolecule P.
Kl :.: [PA] / ] [P] [A]
K2 = [PA21/ [PA] [A]
(2) Kn
=
[PAn1 / [P^n
-l1 [A]where k{Sare the association constants, [P ~ 1 is the concentration of combining molecule P
^i
and [A] is the conc.entration of unbound molecule A.average
associated witheách macromolecule P. This number v ,.is called the average molar number of molecules A interes1:ed infinding the
are we Now
at pH 7.4. The data were ana1yzed in terms of ( 325 )
fatty acids to defatted HSA
mu1tiple stepwise equilibrium. They confirmed that the magnitude of ass'Ociation constants, K, increas as the chain length increas, but the major cooperative bindÎ)1g effects do not occur over the physiological range of fatty acid concentrations.
The magnitude of the first four K's supports the view that the higher energy binding sites of HSA can not be separated 祖todistinct classes.
師大學報
The quantitative ana1ysis of binding nonionic detergents to protein was
第二十三期
studied by Sukow et a1.9-11, using members of the TRITON-X and Igepal-Lo series of nonionic surfactants which differ in their a1kyl structure. They found that the maximum number of nonionic surfactant molecules bound by BSA is pH dependent. At pH 7.0, the extremes of the average molar ratio (
v )
are 5.5are 38 for TRITON X-165 to 6.4 for Lo-530. They also evaluated the equilibrium con-stants and thermodynamic parameters from binding isotherms for TRITON X-114, X-I00, X-I02 and X-165 a1: pH 7.0. In all cases, Scatchard plots are non-linear and suggest cooperative binding for v <2. If the experimental data are for TRITON X-114 to 2.2 for Lo-730, and at pH 2.2 the extremes of
I扭lited to values v
>
2, the Scatchard plots are linear. Equilibrium. association constant in the range of 103 to 104 M-1 were obtained by fitting the data to the stepwise equilibrium model using a leastsquares model fitting procedure.(2) STATEMENT OF PROBLEM AND OBJECfNES
Recent research9 -1 1 has shown that TRITON-X molecules may be used to probe the hydrophobic surfaces of proteins to observe small difference in the binding sites. The average mo1ar ratio
V
of TRITON X-114 molecules bound to albumins shows litt1e vàriation. This suggests that the different animal serumbinding surfaces are remarkably simil訂 in spite of the fact that there is on1y a mechanism, for small variation in am姐o acid composition 1 2. Does the binding
TRITON X-IOO molecules bound to human and another animal serum a1bumin, also reflect minimal evolutionary change1 The evaluation of binding isotherms, association constants
,
and numb.er of binding sites for the TRITON X-IOO* Serum( 329 )