The influence of Al³⁺ ion on porcine pepsin activity in vitro

Figures & data

Figure 1.  Relative activity of pepsin in the presence of Al^{3 +} ions. Proteolytic activity of pepsin expressed as percentage depends on Al^{3 +} ions present. The degree of activation is expressed as % of increased activity considering the activity of pepsin in the absence of Al^{3 +} ions as 100%. All results are expressed as a mean percentage of enzyme activity relative to the corresponding control value, from at least three independent experiments performed in triplicate.

Figure 2.  Lineweaver-Burk plot of a series of kinetics measurements in a presence of different Al^{3 +} ions concentrations at pH2; (inset) Hyperbolic plots (sigmoid fit) representing initial pepsin velocity versus haemoglobin concentration in the absence and presence of different Al^{3 +} ions concentrations at pH2.

Table I.  Influence of Al^{3 +} on K_m, V_max, k_cat and catalytic effectiveness of enzyme (k_cat K_m⁻¹⁾.

[Al^3+] (M)	KS (mM L^− 1)^a	Vmax (μM min^− 1)^a	kcat (min^− 1)^b	kcat KS^− 1 (min^− 1 mM^− 1)
0	0.904 ± 0.083	254 ± 7	2591 ± 70	3264 ± 121
1.7 · 10^− 6	0.907 ± 0.072	332 ± 8	3378 ± 81	3724 ± 126
3.4 · 10^− 5	0.912 ± 0.072	386 ± 10	3938 ± 102	4318 ± 154
1.7 · 10^− 4	0.918 ± 0.073	439 ± 13	4479 ± 132	4879 ± 191
1.7 · 10^− 3	0.921 ± 0.074	497 ± 14	5071 ± 143	5506 ± 209
8.7 · 10^− 3	0.917 ± 0.073	599 ± 17	6112 ± 173	6665 ± 254

^a The K_m and V_max were determined by their respective regression equations; ^b k_cat (turnover number) was calculated using the equation k_cat = V_max/ [E]_t, assuming that the molecular mass of the enzyme is 35 kDa.

Figure 3.  The plots of 1 / (change in slope or intercept), (1/Δ) against 1 / [Al^{3 +} ], where Δ is defined as slope or intercept in the absence of activator (Al^{3 +} ions) minus that in its presence.

Scheme 1 Reaction scheme for non-essential activation; Abbreviations are E—enzyme, S—substrate, A—activator, P—product.

Figure 4.  Activation of pepsin by Al^{3 +} ions; The intercepts on the abscissa of the primary plot of [Hb]/V against reciprocal of activator concentrations provide the values of A₅₀ used to obtain the plots of 1/A₅₀ against v₀/V shown in the inset.

FIG. 5A –UV absorption spectra of pepsin (solid curve) and pepsin treated with 10 mM Al^{3 +} (dotted curve). B - UV absorption spectra of haemoglobin (solid curve) and haemoglobin treated with 10 mM Al^{3 +} (dotted curve). Other spectral curves obtained in a presence of 1 mM and 5 mM Al^{3 +} are not presented because of clarity of the graphs.

FIG. 6  A–IR spectra of pepsin (solid curve,1) and pepsin in a presence of 10 mM Al^{3 +} ions (dotted curve,2) and B - IR spectra of haemoglobin (solid curve,1) and haemoglobin in a presence of 10 mM Al^{3 +} ions (dotted curve,2). IR spectra were recorded in KBr matrices.

Figure 7.  Native PAGE electrophoregram of pepsin (upper) and haemoglobin (lower) without and in a presence of 1, 5 and 10 mM Al^{3 +} at pH 2.