Joint Coordinate Systems

The kinetics of cyanide binding to horse and rabbit a~uomethemoglobins were studied as a function or pll at ionic strength 0.05. 'I'hix study was under-taken to determine the pK values of the "heme-linked"

ionizable groups of these aquomethemoglobins and is an extension of similar work reported earlier (56) on human A and S, pigeon and guinea pi~ aquomethemoglobins.

The kinetics of cyanide binding of three aquomethe-moglobin species have been reported to be monophasic:

the a- and S- chains were found to react at the same rate (O,56). The reaction was monitored under pseudo first order conditions. This was accomplished by reacting

,

aquomethemoglobin with at least a tenfold excess of potassium cyanide in this study. An essential isolation of each of two reacting species can be made by adjusting their

concentrations so that one of them, which is present in conHiderable excess, is effectively maintained at constant concentratjon .

•

For instance, for the reaction,

A + B -+ AB ... (5)

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the rate equation is

-d [A]

dt

=

-d[B]

=

dt ^{k [A] [B] (6 )}

where k is the second order rate constant. If lAl and la] are the initial concentrations and

o ⁰

[A]o is much greater than [B]o' that is, [A]o ~ 10[B]o' the rate equation becomes

-d [A]

dt

=

^-d[B]

=

dt ^{kI [B]} (7)

where kl

=

^{k[A] is the pseudo first order rate constant.}

o

For the above reaction, the rate equation may also be written as:

-d[a-x] ==

dt

where a, is the initial concentration of the reacting species, and x is the amount reacted at time, t.

Since is constant, da

o.

a a

df ⁼⁼

Therefore,

cldxt

=

k ^I [b- x] . •• (8 )

On integration one obtains

•

In b - In[b-x]

=

^{kit • •• (9)}

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In this work the kinetics have been studi~d spectro-photometrically under conditions where the Beer-Lambert law applies. From the Beer-Lambert law, the absorbance of a solution is related to the concentration of the absorbing species by the equation

E

=

^scl

where E is the absorbance, s is the molar absorptivity c is the concentration of absorbing species, and 1 is the path length.

LcL E bu tile absorbance at z(;ro time;

o E the

absor-co

bance at the compeltion of the reaction, and Et the absor-bance at time t after the initiation of the reaction.

'1'11<' following proportionalities hold:

the fraction reacted at time, t, is proportional to

,

Lhc: fraction remaining at Lime t is proportional to

Therefore, e~uation 9 can be expressed in terms of absorbance as:

In[E -E ] - In[E -E ] - k't

o co t ^{co ... (10)}

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73

Since the a and S subunits of aquomethemoglobin react with cyanide ion at the same characteristic rate (49, 56), the following stoichiometric equation describes th~ homogeneous reaction:

Hb+ + CN

kl(app)

HbCN . .. (11)

k_l(app)

In this equation, k1(app) is the apparent second order combination rate constant and k_1(app) is the apparent first order dissociation rate constant.

Under pseudo first-order conditions, the observed rate constant, k b ' is related to the apparent rate

o s

constants by the equation

••• (12)

,

Figure 1 shows, for rabbit aquomethemoglobin, typical plots of -In (Et-Ero] against time (c.f. Equation 10) at various fixed cyanide concentrations at a given pH. The plots are monophasic; similar plots were obtained for horse aquomethemoglobin (not shown). These monophasic plots strongly indicate that the a- and S- subunits

are kinetically

•

equivalent with respect to cyanide binding, as has been reported for other aquomethemoglobins (56) .

•

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28

o

1.4oL---20"'---I..-- ..•...6o~-~80~-~100~-~12~O---:17;"O::----:-:160~

TIME (See) FIG. 1.

Pseudo first-order rate plots for tile binding of cyanide ion to rabbit aquomethemoglobin. Conditions: Phosphate buffer, pH

=

6.2; ionic strength

=

^{0.05 M; ~OoC [MetHb]}

-2 ]J~1heme. The KCN concentrations are 30]JM [filled circles], 60 pM (open circles) and 80 pM (squares) See Appendix 1

Table 6~

•

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35

5

(CW) 6~ 90 ₁₂₀

'I 1

if)

x 1

11o ::x::

FIG. 2.

Dependence of observed rate constant, k b ' on cyanide o s

concentration at 200C for rabbit aquomethemoglobin ph

=

^6.2

[filled circles], pH

=

7.0 [open circles] anu pH

=

^{8.6 [Square~}

See Appendix 1 Table 8.

,

•

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•

70

p~

8.0 9.0

6.0

FIG 3.

-Dependence of the apparent second order rate constant for cyQnidc binding to aquomethemoglobin, kapp' on pH at 200C I = 0.05. These art theoretical lines obtained from the computer. Human A [filled squares], Horse [open circles], and Rabbit [filled circles]. See Appendix I Tables 9 ana 10 for horse and rabbit aquomethemoglobin respectively.

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'I'ho s-IoJws of these plots p;i ve k I. ' the ohserved

O)S

pseudo fil':::;t-orderrate c onst.ant for cyanide bindin g.

The ~traight line plots also indicate that the reaction is strictly ps(>udo first-order under the experimental conaitions employed in this work. A linear least squares regression program was used to calculate _{k b .}

o s Kinetic runs were carried out three times under the same experi-mental condjtions. Values of mean k b for individual

o s

runs are listed in Tables 7 and 8 for horse and rabbit aquomethemoglobins, respectively [See Appendix 1 pages 157-159]. The intercepts in Fig. 1 are not equal but it is due to experimental error which does not affect our experimental data. Figure 2 shows plots of k b versus

o s

cyanide concentrations at fixed pH. Similar plots were ohtaincd for horse aquomethemoglobjn (not shown). As

expect~d from equation 12, the plots are linear, Values of k1(app) were obtained from the slopes by least squares regression program. It is known from the results of Job et al (54) that K 1( ) values are very small and their

- app

determination from the intercepts of plots according to

equation 12 would be subject to a great deal of uncertainty.

No attempt "'astherefore made to evaluate k ( ).

-1 app Figure 3 shows plots of k1(app) ap;ainst BH for horse and rabbit aquornethemoglobins. The plot for human A aquomethemoglobin (56) is included for comparison. Tables

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9 and 10 (see Appendix 1 Pages ICI -162 ) list the values of k1(app) for horse and rabbit aquomethemoglobin, respectively at various pH values.

The pH profiles for horse and rabbit aquomethemoglobins are similar to those of other aquomethemoglobins (56).

Such pH dependence data have been satisfactorily accounted for with Scheme 1(56).

SCHEME ₁

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