- Email: [email protected]
Kinetic comparison of human, canine and porcine pepsins
Comp. Biochem. Physiol., 1971, 1Iol. 39B,pp. 715 to 718. Pergamon Press. Printed in Great Britain
KINETIC COMPARISON OF HUMAN, CANINE AND PORCINE PEPSINS G. L. T R I T S C H and C. R. S A C H A T E L L O * Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203 (Received 8 January 1971) Abstract--1. The variation of the Michaelis constant (K,,) and the maximum
reaction velocity (V) with pH for the reaction of hemoglobin with human, canine and porcine pepsins was studied. 2. At pH 2 the K~'s were found to be 6"3 x 10 -4 M, 10x 10 -4 M and 1"1 x 10 -4 M, respectively, for human, canine and porcine pepsins. 3. The similarity between human and canine pepsins permits valid comparisons of these enzyme activities in molar terms, and porcine pepsin appears to be a reasonable gravimetric standard for such studies. INTRODUCTION THE DOG has been used widely as a model for the study of human gastrointestinal physiology (Emas et al., 1967). Although the secretion of acid by the dog has been intensively studied and compared to man, relatively little is known of human and canine pepsins. Porcine pepsin is the only one available commercially in crystalline form and has been used in most chemical studies of this enzyme (Dixon & Webb, 1964). In order to be able to express pepsin secretion in molar terms in dogs and men, it is necessary to use a homologous isoenzyme of known molecular weight as the standard. T h e conclusion that equivalent proteolysis is attributable to equal numbers of enzyme molecules of two such enzymes is valid only if the Michaelis constants (Kin) are similar at the pH of the experiment. Although crystalline porcine pepsin has been found to be heterogeneous (Ragagopalan et al., 1966), its molecular weight is known to be about 34,000 and the various components are active in hemoglobin proteolysis. Reported herein is the variation of the Michaelis constant (K,,) and the extrapolated maximum rate of reaction at infinite substrate concentration (V) with pH for hemoglobin of crystalline porcine pepsin and human and canine gastric juices. MATERIALS AND METHODS Proteolysis was determined in buffers consisting of 0"1 M citric acid adjusted to the desired pH at 37°C with NaOH. Stock solution of twice-crystaUized human hemoglobin (Pentex) of 20 mg/ml (3 x 10 -4 M) were prepared in the buffer of appropriate pH and ten assays with substrate concentrations between 0"1 and 3"0 x 10 -4 M performed at each pH. The amount of enzyme was such that almost maximum reaction velocity would be achieved * Present address: Department of Surgery, University of Kentucky Medical Center, Lexington, Kentucky. 715
716
G . L . TRITSCH AND C. R. SACHATELLO
at the highest substrate concentration. To 1'0 ml of buffered hemoglobin solution was added 0"1 ml of enzyme solution and incubated at 37°C for 10 min. Two ml of 10°o trichloroacetic acid was then added, the precipitate removed by centrifugation and the absorbance at 280 m/, of the supernatant determined in 1-cm light path cells. The velocity of the reaction, v, was defined as the net increase in absorbance observed during this incubation. The results at each pH were analyzed by the Hanes (1932), Lineweaver-Burke (1934) and Eadie (1942) plots to give the best estimate of Km and V. Although these plots have different inherent validity (Dowd & Riggs, 1965), good agreement in the extrapolated values for Km and V was found. The average of the data from these plots is plotted against pH as suggested by Dixon & Webb (1964) and shown in Fig. l. In this plot the points are connected by straight lines of integral slope, i.e. zero, one unit, two units, positive or negative, and an upward inflection point with increasing pH indicates the p K of a group situated on the enzyme-substrate complex. (a) I
~
u
i
log V
i
-
o
(b)
I
4b. ~ . pKm 3 ~
I
-0og
I t I
--
rl2
i! pH
FIC. 1. Dixon plot of hemoglobin proteolysis by human, canine and porcine pepsins. The straight lines of one unit or zero slopes were fitted by eye. a. Log of maximum velocity (V) of human (©), canine ([~) and porcine (Q) pepsin plotted against pH. b. Negative logarithm of Michaelis constant (K~) plotted against pH. It can be seen that the inflection points in the plots (indicated by the vertical broken lines) for human and canine pepsin are the same, indicating similarity in the dissociation constant of a group on the enzyme-substrate complex. Crystalline porcine pepsin was obtained from Worthington, (Freehold, N.J., U.S.A.) canine gastric juice was collected from unstimulated female mongrel dogs (10 kg) with a stainless-steel gastric fistula, and human gastric juice from overnight secretions of patients subsequently determined to be free of gastrointestinal disease. RESULTS AND DISCUSSION T h e data in Fig. 1 s h o w the Km's for h u m a n and canine pepsin for h e m o g l o b i n to be similar at the low p H of gastric juice, i.e. 6.3 and 10.0 x 10 .4 M , respectively at p H 2. Porcine pepsin has a lower K m of 1.1 x 10 .4 M. A frequently used rule of t h u m b is that two e n z y m e s are very similar or even alike if the Km's are within one order of magnitude. T h e inflection points of the D i x o n plots confirm this finding.
K I N E T I C COMPARISON OF HUMAN, CANINE AND P O R C I N E PEPSINS
717
The ionizing group on the enzyme--substrate complexes of human and canine pepsin-hemoglobin show the same dissociation constant (pK = 3.0) while that for porcine pepsin-hemoglobin is at a lower pH. The log V vs. pH plot confirms these findings. These similarities among the pepsins from the three species herein tested justify the comparison of these enzymes in terms of moles of pepsin responsible for a given degree of proteolysis of human hemoglobin. Since there are many peptide bonds in hemoglobin susceptible to pepsin, and it seems unlikely that the same bonds are cleaved in the same sequence by pepsins from the three species, it might be argued that synthetic peptide substrates might have been preferable substrates for this study. However, gastric juice and purified pepsin undergo rapid autolysis at low pH and the several fragments obtained, while capable of proteolysis of hemoglobin, all will not attack the synthetic substrates. Furthermore, the Km for these substrates is about one order of magnitude larger than for hemoglobin (Bergmeyer, 1963). The higher specificity for hemoglobin, therefore, makes the latter a preferable substrate. Bovine hemoglobin powder, which is cheaper than the human protein and has been used in many studies, is not completely soluble at low pH, which makes the substrate concentration variable and uncertain. In view of the Km's herein observed, the substrate concentration of 0.1-3 x 10 -4 M is lower than would be proposed for routine assays. However, substrate inhibition was observed at high hemoglobin concentrations at pH 1.5-2, and 3 × 10 -4 M was near the maximum solubility of crystalline human hemoglobin at 37°C at pH 3-5 and above. The validity of the observed reaction velocities was confirmed by obtaining equivalent values for K m and V with both 50% lower enzyme concentrations and with shorter and longer incubation periods. It is concluded that the similarity of human and canine pepsins permits valid comparisons to be made of these enzymes on a molar basis at pH 2 and that porcine pepsin is a reasonable gravimetric standard for such studies. Acknowledgement--This investigation was carried out with the very capable assistance of Janice Walter.
REFERENCES BERGMEYERH. U. (1963) Methods of Enzymatic Analysis, p. 990. Academic Press, New York. DIXON M. & WEBB E. C. (1964) Enzymes, 2nd edn, pp. 128-145. Academic Press, New York. Dowv J. E. & RIoos D. S. (1965) A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations..7, biol. Chem. 240, 863-869. EADIEG. S. (1942) The inhibition of cholinesterase by phytostigmine and prostigmine..7. biol. Chem. 146, 85-93. EMAS S., SWANK. G. & JACOBSONE. D. (1967) Methods of studying gastric secretion. In Handbook of Physiology, Section 6, Volume II, "Alimentary Canal" (Edited by CODEC. F.), Chapter 42. Am. Physiol. Sot., Washington, D.C. HnrcEs C. S. (1932) Studies of plant amylases. Biochem.ff. 26, 1406-1421.
718
G . L. TRITSCH AND C. R. SACHATELLO
LINEWI~AVERH. & BURK D. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56, 658-667. RAGACOP~a.ANT. G., MOORE S. & STEINW. H. (1966) Pepsin from pepsinogen; preparation and properties, ft. biol. Chem. 241, 4940--4950. Key Word Index--Human pepsin; canine pepsin; porcine pepsin; hemoglobin ; enzyme kinetics; gastric juice; Michaelis constant.
KINETIC COMPARISON OF HUMAN, CANINE AND PORCINE PEPSINS G. L. T R I T S C H and C. R. S A C H A T E L L O * Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203 (Received 8 January 1971) Abstract--1. The variation of the Michaelis constant (K,,) and the maximum
reaction velocity (V) with pH for the reaction of hemoglobin with human, canine and porcine pepsins was studied. 2. At pH 2 the K~'s were found to be 6"3 x 10 -4 M, 10x 10 -4 M and 1"1 x 10 -4 M, respectively, for human, canine and porcine pepsins. 3. The similarity between human and canine pepsins permits valid comparisons of these enzyme activities in molar terms, and porcine pepsin appears to be a reasonable gravimetric standard for such studies. INTRODUCTION THE DOG has been used widely as a model for the study of human gastrointestinal physiology (Emas et al., 1967). Although the secretion of acid by the dog has been intensively studied and compared to man, relatively little is known of human and canine pepsins. Porcine pepsin is the only one available commercially in crystalline form and has been used in most chemical studies of this enzyme (Dixon & Webb, 1964). In order to be able to express pepsin secretion in molar terms in dogs and men, it is necessary to use a homologous isoenzyme of known molecular weight as the standard. T h e conclusion that equivalent proteolysis is attributable to equal numbers of enzyme molecules of two such enzymes is valid only if the Michaelis constants (Kin) are similar at the pH of the experiment. Although crystalline porcine pepsin has been found to be heterogeneous (Ragagopalan et al., 1966), its molecular weight is known to be about 34,000 and the various components are active in hemoglobin proteolysis. Reported herein is the variation of the Michaelis constant (K,,) and the extrapolated maximum rate of reaction at infinite substrate concentration (V) with pH for hemoglobin of crystalline porcine pepsin and human and canine gastric juices. MATERIALS AND METHODS Proteolysis was determined in buffers consisting of 0"1 M citric acid adjusted to the desired pH at 37°C with NaOH. Stock solution of twice-crystaUized human hemoglobin (Pentex) of 20 mg/ml (3 x 10 -4 M) were prepared in the buffer of appropriate pH and ten assays with substrate concentrations between 0"1 and 3"0 x 10 -4 M performed at each pH. The amount of enzyme was such that almost maximum reaction velocity would be achieved * Present address: Department of Surgery, University of Kentucky Medical Center, Lexington, Kentucky. 715
716
G . L . TRITSCH AND C. R. SACHATELLO
at the highest substrate concentration. To 1'0 ml of buffered hemoglobin solution was added 0"1 ml of enzyme solution and incubated at 37°C for 10 min. Two ml of 10°o trichloroacetic acid was then added, the precipitate removed by centrifugation and the absorbance at 280 m/, of the supernatant determined in 1-cm light path cells. The velocity of the reaction, v, was defined as the net increase in absorbance observed during this incubation. The results at each pH were analyzed by the Hanes (1932), Lineweaver-Burke (1934) and Eadie (1942) plots to give the best estimate of Km and V. Although these plots have different inherent validity (Dowd & Riggs, 1965), good agreement in the extrapolated values for Km and V was found. The average of the data from these plots is plotted against pH as suggested by Dixon & Webb (1964) and shown in Fig. l. In this plot the points are connected by straight lines of integral slope, i.e. zero, one unit, two units, positive or negative, and an upward inflection point with increasing pH indicates the p K of a group situated on the enzyme-substrate complex. (a) I
~
u
i
log V
i
-
o
(b)
I
4b. ~ . pKm 3 ~
I
-0og
I t I
--
rl2
i! pH
FIC. 1. Dixon plot of hemoglobin proteolysis by human, canine and porcine pepsins. The straight lines of one unit or zero slopes were fitted by eye. a. Log of maximum velocity (V) of human (©), canine ([~) and porcine (Q) pepsin plotted against pH. b. Negative logarithm of Michaelis constant (K~) plotted against pH. It can be seen that the inflection points in the plots (indicated by the vertical broken lines) for human and canine pepsin are the same, indicating similarity in the dissociation constant of a group on the enzyme-substrate complex. Crystalline porcine pepsin was obtained from Worthington, (Freehold, N.J., U.S.A.) canine gastric juice was collected from unstimulated female mongrel dogs (10 kg) with a stainless-steel gastric fistula, and human gastric juice from overnight secretions of patients subsequently determined to be free of gastrointestinal disease. RESULTS AND DISCUSSION T h e data in Fig. 1 s h o w the Km's for h u m a n and canine pepsin for h e m o g l o b i n to be similar at the low p H of gastric juice, i.e. 6.3 and 10.0 x 10 .4 M , respectively at p H 2. Porcine pepsin has a lower K m of 1.1 x 10 .4 M. A frequently used rule of t h u m b is that two e n z y m e s are very similar or even alike if the Km's are within one order of magnitude. T h e inflection points of the D i x o n plots confirm this finding.
K I N E T I C COMPARISON OF HUMAN, CANINE AND P O R C I N E PEPSINS
717
The ionizing group on the enzyme--substrate complexes of human and canine pepsin-hemoglobin show the same dissociation constant (pK = 3.0) while that for porcine pepsin-hemoglobin is at a lower pH. The log V vs. pH plot confirms these findings. These similarities among the pepsins from the three species herein tested justify the comparison of these enzymes in terms of moles of pepsin responsible for a given degree of proteolysis of human hemoglobin. Since there are many peptide bonds in hemoglobin susceptible to pepsin, and it seems unlikely that the same bonds are cleaved in the same sequence by pepsins from the three species, it might be argued that synthetic peptide substrates might have been preferable substrates for this study. However, gastric juice and purified pepsin undergo rapid autolysis at low pH and the several fragments obtained, while capable of proteolysis of hemoglobin, all will not attack the synthetic substrates. Furthermore, the Km for these substrates is about one order of magnitude larger than for hemoglobin (Bergmeyer, 1963). The higher specificity for hemoglobin, therefore, makes the latter a preferable substrate. Bovine hemoglobin powder, which is cheaper than the human protein and has been used in many studies, is not completely soluble at low pH, which makes the substrate concentration variable and uncertain. In view of the Km's herein observed, the substrate concentration of 0.1-3 x 10 -4 M is lower than would be proposed for routine assays. However, substrate inhibition was observed at high hemoglobin concentrations at pH 1.5-2, and 3 × 10 -4 M was near the maximum solubility of crystalline human hemoglobin at 37°C at pH 3-5 and above. The validity of the observed reaction velocities was confirmed by obtaining equivalent values for K m and V with both 50% lower enzyme concentrations and with shorter and longer incubation periods. It is concluded that the similarity of human and canine pepsins permits valid comparisons to be made of these enzymes on a molar basis at pH 2 and that porcine pepsin is a reasonable gravimetric standard for such studies. Acknowledgement--This investigation was carried out with the very capable assistance of Janice Walter.
REFERENCES BERGMEYERH. U. (1963) Methods of Enzymatic Analysis, p. 990. Academic Press, New York. DIXON M. & WEBB E. C. (1964) Enzymes, 2nd edn, pp. 128-145. Academic Press, New York. Dowv J. E. & RIoos D. S. (1965) A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations..7, biol. Chem. 240, 863-869. EADIEG. S. (1942) The inhibition of cholinesterase by phytostigmine and prostigmine..7. biol. Chem. 146, 85-93. EMAS S., SWANK. G. & JACOBSONE. D. (1967) Methods of studying gastric secretion. In Handbook of Physiology, Section 6, Volume II, "Alimentary Canal" (Edited by CODEC. F.), Chapter 42. Am. Physiol. Sot., Washington, D.C. HnrcEs C. S. (1932) Studies of plant amylases. Biochem.ff. 26, 1406-1421.
718
G . L. TRITSCH AND C. R. SACHATELLO
LINEWI~AVERH. & BURK D. (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc., 56, 658-667. RAGACOP~a.ANT. G., MOORE S. & STEINW. H. (1966) Pepsin from pepsinogen; preparation and properties, ft. biol. Chem. 241, 4940--4950. Key Word Index--Human pepsin; canine pepsin; porcine pepsin; hemoglobin ; enzyme kinetics; gastric juice; Michaelis constant.