In vitro Simulation of Gastric and

Pancreatic Phase Digestion of Meat

and Bone Meal: Studies of Pepsin Autolysis

Y. Qiao and T. van Kempen

Summary

It was demonstrated that the deactivation of pepsin took the form of autolysis. The more unfavorable the condition (less favorable pH, higher concentration), the less autolysis and the more denaturation occurred.

The autolysis of pepsin was faster at more favorable pH (pH 2.0 versus pH 4.0). The higher the concentration, the slower the autolysis was. Both pH and concentration had significant effects on the maximal degree of autolysis. However, the degree of autolysis varied within a narrow range. On average, the maximal degree of autolysis was 11.0 %, and pepsin autolysate peptide contained 9.1 amino acid residual. The autolysis of pepsin at 0.5 mg/ml in pH 4.0 buffer became sigmoidal rather than exponential. No substrate protection was found.

Introduction

Proteolytic enzymes have a unique feature. Under biological conditions, such an enzyme molecule will digest other enzyme molecules of the same nature as if they are substrate. This hydrolytic process is referred to as autolysis. In the field of in vitro digestion of feed proteins, most researchers have neglected the contamination of enzyme autolysates on the measurement of amino acid. This negligence of autolysis should result in overestimation of digestible amino acids, particularly when relatively a large quantity of enzymes is used.

In our study of in vitro digestion of meat and bone meal (MBM), the hydrolysis of MBM protein was measured by o-pthalaldehyde (OPA). In order to obtain the true hydrolysis of MBM, the hydrolysis of digestive proteases needs to be estimated. We therefore studied the autolysis of pepsin (PP).

Materials and Methods

Porcine pepsin (PP) was used for incubation in acidic citrate buffer solutions (CBS, pH 2.0 or pH 4.0). The buffer contained 0.06% sodium azide to prevent microbial fermentation.

All incubation test tubes were fitted into a revolving plate to facilitate the contact between enzyme molecules and enzyme-MBM proteins. The incubation was done at 38 C. For measurement of degree of autolysis, at different time intervals, a small aliquot of the digesta was pipetted out and mixed with equal volume of 20% sodium dodecyl sulfate (SDS) to denature the enzyme. After centrifugation at 14,000 g for 5 minutes, the supernatant was used for analysis with the OPA technique. For measurement of enzyme activity decay, at time intervals, a small aliquot (usually 5-50 microliters) was taken out from the incubation and the activity was measured immediately. Enzyme activity was expressed in unit. One unit was defined as an increase of 0.001 units of absorbance per minute per mg of enzyme.

Experiment 1: The decay of pepsin activity in CBS without MBM

Pepsin at different concentrations was incubated in 0.05 M CBS at pH 2.0 or pH4.0. At time intervals, the activity of pepsin was measured using the hemoglobin method. Data were analyzed with the model [Et]=[Ei]*(1-exp (-kd*t)). The rate of decay (kd) and the half-life (t _½) were calculated. The results are presented in the Table 1.

Table 1. The rate of pepsin activity decay at different concentration and pH.

	CBS pH 2.0		CBS pH 4.0
Pepsin (mg/ml)	kd^1(1/hr)	t ½ (hrs)	kd^1(1/hr)	t ½ (hrs)
0.05	0.2400^a	2.9	0.1536^a	4.5
0.10	0.1675^a	4.2	0.0963^b	7.2
0.20	0.0850^b	8.2	0.0732^b	9.5
0.50	ND^2	ND^2	NA^3	14.4
^1Values were obtained from nonlinear regression of 2 replicates. Different superscripts imply statistical significance calculated from the asymptotic standard error (p<0.05). ^2 Not done. ^3 Not applicable because the exponential decay did not suit the data.

The results showed that pH and concentration had significant effects on the rate of activity loss. At more favorable pH, the rate of activity loss was faster. The higher the concentration, the slower the rate of activity loss was.

Experiment 2: The degree of autolysis of pepsin without MBM

The peptide bond released from pepsin was measured using the OPA technique. The data were analyzed with the model [Penzyme]=[Ei](1-exp(-ka*t)), where [Penzyme] is the product from autolysis, [Ei] is the intial peptide bond concentration, ka is the rate of autolysis, t is time of sampling. The degree of autolysis (DA) was calculated using DA=[Penzyme]/[Ei]=c*(1-exp(-ka*t)). Table 2 shows the rate of autolysis (ka) and the time (t _½) needed to reach 50% of the maximal degree of autolysis (c).

Table 2. The rate of autolysis (ka) of pepsin at different concentration and pH.

	CBS pH 2.0		CBS pH 4.0
Pepsin (mg/ml)	kd^1(1/hr)	t ½ (hrs)	kd^1(1/hr)	t ½ (hrs)
0.05	0.1946^ab	3.6	0.1099^bc	6.3
0.10	0.1951^ad	3.6	0.0703^bce	9.9
0.20	0.1213^b	5.7	0.0636^e	10.9
^1Values were obtained from nonlinear regression of 2 replicates. Different superscripts imply statistical significance calculated from the asymptotic standard error (p<0.05).

The results showed that the pH and concentration had significant effects on the rate of peptide bond breakdown. At more favorable pH, the release of peptide bonds was faster. The higher the concentration, the slower the rate of peptide bonds breakdown.

The predicted maximal degree of autolysis (c) as obtained from nonlinear regressions, are presented in Table 3.

Table 3. The maximal degree of autolysis (c) of pepsin at different concentration and buffer pH.

	c^1 (%)
Pepsin (mg/ml)	CBS pH 2.0	CBS pH 4.0
0.05	9.2^a	10.5^ace
0.10	11.7^bc	10.^ac
0.20	11.8^bd	9.6^a
0.50	ND^2	14.2^f
^1Values were obtained from nonlinear regression of 3 replicates. Different superscripts imply statistical significance calculated from the asymptotic standard error (p<0.05). ^2Not done.

It was obvious that although there was an effect of pH and an effect of pepsin concentration on the rate of activity decay, neither pH nor concentration had greatly changed the maximal degree of autolysis of pepsin. On the whole, the average maximal degree of autolysis was around 11.0%. This implied that the average peptide length was 9.1 amino acid residual when maximal autolysis of pepsin was reached.

Experiment 3. Sigmoidal deactivation of pepsin

At high concentration (0.5 mg/ml, pH 4.0), the decay of pepsin activity became sigmoidal rather than exponential. Figure 1 shows the activity decay of pepsin at 0.5 mg/ml at pH 4.0.

Figure 1. Sigmoidal decay of pepsin activity at higher concentration of 0.5 mg/ml in 0.05 M CBS pH 4.0 at 38 C. 3 replicates at each time point. The pepsin activity was measured using the hemoglobin method at 25 C. A sigmoidal model (Et=Ei(1- t²/(a+b t²)) was used for regression. Estimation was Ei=1612, a=249.4, b=0.7977. The half life t_½ was 14.4 hours.

The pattern of production of free amino group from peptide bonds from pepsin incubated at 0.5 mg/ml, 0.05 M CBS pH 4.0 also changed to sigmoidal, in agreement with the activity decay under identical conditions (Figure 2).

Figure 2. Sigmoidal increase of degree of autolysis (DA) of pepsin at 0.5 mg/ml in 0.05 M CBS pH 4.0 at 38 C. 3 replicates at each time point. DA=[Penzyme]/[Ei]. Penzyme was measured using the OPA method. A sigmoidal model (DA= t²/(a+b t²) was used for regression. Estimation was a=1953.9, b=6.6674. The time needed to achieve 50% of maximal DA (c) was t_½ =16.2 hours.

The activity decay and release of peptide bonds both showed a sigmoidal pattern when pepsin concentration was at 0.5 mg/ml and incubated at pH 4.0. This supported the thinking that the pattern of release of peptide bond from pepsin was correlated to the concentration.

In any circumstances, the loss of activity was correlated to the breakdown of peptide bonds. It is proposed that the loss of pepsin activity took more form of autolysis rather than pure denaturation.

It is speculated that at higher concentration, two pepsin molecules have larger chance to bind each other by the active sites. The binding, which can be expressed as active site to active site binding, prevented the autolysis of any molecules. At lower concentration, this chance was reduced and the chance that binding of one molecule acting as enzyme to the other molecule as substrate was increased. Thus the half-life of the enzyme was shortened.

Experiment 4: The decay of pepsin activity in the presence of MBM protein

pepsin (0.125 mg/ml) was incubated with MBM (12.5mg protein/ml) at 38 C in CBS pH 2.0. At different hours after inoculation, an aliquot was taken for measurement of pepsin activity using hemoglobin (Hb) as substrate at room temperature (25 C).

The results are summarized in Figure 3.

Figure 3. Pepsin (0.125 mg/ml) activity Decay. Results of 3 replicates. The line was the predicted values by nonlinear regression using the model [Et]=[Ei]*(1-exp(-kd*t)). [Et] is the activity at time of measurement, [Ei] is the initial activity. The half life t_½ was estimated to be 3.3 hours.

Pepsin activity was not extended longer than a day. Actually, by 12 hours post inoculation, pepsin was nearly inactive. The half-life of pepsin at around 0.1 mg/ml with or without MBM was 3.3 and 3.6 hours, respectively. This suggested that the substrate did not protect pepsin.