Alcohol, Vol.9, pp. 181-184, 1992
0741-8329/92 $5.00 + .00 Copyright©1992PergamonPress Ltd.
Printed in the U.S.A. All rightsreserved.
Acetaldehyde Decreases the Antitryptic Activity of a -Proteinase Inhibitor ARTHUR
S. B R E C H E R 1 A N D J E N N I F E R
L. P A V L O C K
Department o f Chemistry, Bowling Green State University, Bowling Green, OH 43403 Received 20 S e p t e m b e r 1991; A c c e p t e d 6 D e c e m b e r 1991 BRECHER, A. S. AND PAVLOCK J. L. Acetaldehydedecreasesthe antitrypticactivity of arproteinase inhibitor. ALCOHOL 9(3) 181-184, 1992.-The trypsin-inhibiting activity of human serum is lowered upon addition of formaldehyde or acetaldehyde thereto. Acetaldehyde reacts with a-m-proteinase inhibitor (arPI) to decrease its trypsin-inhibiting ability. Acetaldehyde has only a slight effect on the tryptic hydrolysis of benzoyi-DL-arginine-p-nitroanilide. It did not decrease the inhibitory activity of the Kunitz inhibitor (Aprotinin) or soybean trypsin inhibitor. Since aldehydes form covalent products with primary amines, primary amides, arginine, tyrosine, and tryptophan in protein, as well as methylene bridges thereby crosslinking functional groups, it is proposed that one or more such interactions affect at-PI activity. It is further suggested that chronically high levels of acetaldehyde, as a metabolic product of ethanol, may be a contributory factor to the generation of pancreatitis in alcoholics by possibly lowering the effective cq-PI level which is a natural protective element from proteolysis by trypsin. Trypsin
aj-Proteinase inhibitor
Acetaldehyde
Human serum
Alcohol
Alcoholism
Pancreatitis
In view of the observation of pancreatitis in some alcoholics afflicted with cirrhosis, and in view of the natural metabolic transformation of ethanol to acetaldehyde, it was hypothesized that acetaldehyde may react with naturally occurring trypsin inhibitors in liver and serum, thereby causing physiological inactivation of the inhibitors. It was further hypothesized that trypsin-like activity would be elevated as a result of lowered antitrypsin activity, thereby promoting the development of pancreatitis. This communication reports on the lowering of trypsin-inhibiting activity of human serum upon treatment of the serum with acetaldehyde or formaldehyde, as well as the reduction of the trypsin-inhibiting activity of a~-proteinase inhibitor (,~rPI) upon preincubation with acetaldehyde, supporting the suggestion that chemical modification of trypsin inhibitors, in addition to genetic factors (15) and impairment of protein synthesis (1), might influence the onset of pancreatitis.
ACUTE pancreatitis is a condition which is characterized, in part, by an increase in pancreatic enzymes in the serum. Among the enzymes observed to be elevated are amylase, lipase, and a trypsin-like activity (15). As a result of activation of trypsin and other proteases, digestion of the pancreas and peripancreatic tissues is observed. One of the principle causes of pancreatitis in the Western world is the consumption of alcoholic beverages, although numerous other causes have been implicated (15). Alcohol uptake by humans is a major factor in the generation of cirrhosis of the fiver (1). Pediatric arantitrypsin deficiency may also induce cirrhosis of the liver (1). Ethanol uptake causes morphological and functional changes in the hepatocytes. It is metabolized to acetaldehyde which may react with cellular components in the hepatocyte and elsewhere since it circulates freely (1). Aldehydes are highly reactive. They are reported to react with proteins (10,17), thereby affecting conformation and function. Aldehydes are utilized to denature nucleic acids by reaction with amino functional groups and ring nitrogen (14,16,18), as observed with adenine, guanine, uracil, and cytosine in nucleic acids (18,20). Various amino acids in proteins also form covalent products with aldehydes. Primary amines, primary amides, guanidyl groups, indole groups, and phenolic groups are susceptible to modification by formaldehyde leading in some cases to crosslinking (5-9). Modification of ribonuclease A by formaldehyde results in a 40% reduction in enzymic activity within 2 minutes (17), and further reduction in activity with longer exposure of the enzyme to the aldehyde.
EXPERIMENTAL Trypsin, 2 x crystalline, was purchased from Nutritional Biochemical Corp. (Cleveland, OH). Benzoyl-DL-arginine-pnitroanilide (BAPA) and soybean trypsin inhibitor (SBTI) were obtained from Mann Research Laboratories (New York, NY). t~rPI and aprotinin, the basic Kunitz inhibitor, were purchased from Sigma Chemical Co. (St. Louis, MO). AG501-X8 mixed bed resin was supplied by Bio-Rad (Richmond, CA).
J To whom reprint requests should be addressed. 181
182
BRECHER AND PAVLOCK
o.41
(A)
(B)
@.6
o.E =-
'E 0.4
e-
==
~ 0.2
O L..
=. G-J
,~
&
R
/ 0.1
0.0
Control
Serum
.~rum + f (0.29 M)
0.0
~ (1.16 M)
Control
Serum
Serum ÷ (0.19 M) (0.75 M)
(1.5 M)
FIG. 1. Effect of formaldehyde on the trypsin-inhibiting ability of human serum (mean + SEM). (A) To 25-/zl aliquots of human serum in capped Eppendorf tubes were added 0, 2, and 8/zl of 37% formalin and sufficient 0.2 M Pi (Na÷), pH 8.0, to give of total volume of 80/xl. The formaldehyde concentrations were 0.29 and 1.16 M, respectively. The mixture was preincubated at room temperature for 1 hour, after which time 20 t~l of trypsin (20/xg in 1 mM HC1) were added. A control trypsin without serum or formaldehyde was also prepared. After further incubation at room temperature for 30 minutes, aliquots of 10/zl were added to 5 ml of buffered substrate and 990/A of dH20. The solutions were incubated at 370C for 30 minutes, and the reaction was stopped by the addition of 1 ml of 30% acetic acid. The release of p-nitroaniline was quantitated by measuring A4~0(n = 4). (The formaldehyde concentrations in the incubation mixtures were 3.8 x 10 -4 M and 1.5 x 10 -3 M, respectively.) (B) Effect of acetaldehyde upon the trypsin-inhibiting ability of human serum (mean + SEM). To 25-/A aliquots of human serum in capped Eppendorf tubes were added 2, 8, and 16/zl of resin-treated 33% acetaldehyde and sufficient 0.2 M Pi (Na+), pH 8.0, to a total volume of 80/~1. The acetaldehyde concentrations were 0.19 M, 0.75 M, and 1.5 M, respectively. The mixture was preincubated at room temperature for 1 hour, after which time 20 #1 of trypsin (20/zg in 1 mM HC1) were added. A trypsin control was also prepared. After standing an additional 30 minutes at room temperature, 10/zl of the mixtures were taken for assay as described for (A) (n = 4). (The acetaldehyde concentrations in the incubation mixtures were 2.5 × 10 -4 M, 1 × 10 -4 M, and 2 × I0 -3 M, respectively.)
Trypsin activity was quantitated by the procedure o f Erlanger et al. (3) which measures the release o f p-nitroaniline from benzoyl-DL-arginine-p-nitroaniline'HC1 (BAPA). B A P A (43.5 mg) was taken up in 1 ml dimethylsulfoxide, and the solution was diluted to 100 ml with 0.05 M Tris/HC1, p H 8.2/0.02 M CaC12. To 5 ml o f buffered substrate was added 20/~l trypsin (1 m g / m l in 1 m M HCI) and sufficient distilled H20 to a total o f 6 ml. The solution was incubated in a shaking water bath at 37°C for 30 or 60 rain, as indicated in the Fig. 1 legend, after which time the reaction was terminated by the addition o f 1 ml of 30% acetic acid. The p-nitroaniline generated during the course of the reaction was determined by reading the absorption at 410 nm. Conditions for studying the effect of serum, formaldehyde, acetaldehyde, and tx~-PI on tryptic activity are given in the Figure legends. Aliquots o f formaldehyde and acetaldehyde were passed through short
columns of AG-501-X8 mixed bed resin in order to remove oxidation products (formic and acetic acids). (It was observed, however, that formaldehyde and acetaldehyde exhibited the same effect on the proteins with or without exposure to the resin.) Acetaldehyde gave a negative response for the presence of peroxides with the potassium idodide reaction and upon reaction with ferrous a m m o n i u m sulfate and potassium thiocyanate. RESULTS The data in Fig. 1 reflect the effect of aldehydes upon the ability o f human serum to inhibit the tryptic hydrolysis of B A P A . Figure 1A indicates the effect o f preincubation of serum at r o o m temperature with formaldehyde upon its capacity to inhibit tryptic activity. Tryptic activity in formalde-
ALDEHYDES INACTIVE aI-PROTEINASE INHIBITOR
183
hyde-treated serum is essentially tripled, as compared with its activity in an untreated serum mixture. Results are comparable when formaldehyde is passed through a Bio-Rad AG-501X8 mixed bed resin to remove oxidation products (data not shown). Figure IB shows that treatment of serum with resintreated acetaldehyde similarly decreases the trypsin-inhibiting capacity of the serum. Acetaldehyde, of itself, has only a very slight effect upon tryptic activity. Since cq-PI is the major trypsin inhibitor in serum, the effect of acetaldehyde upon its physiological effect was examined. The data in Fig. 2 indicate that pretreatment of cq-PI with acetaldehyde considerably reduces the trypsin-inhibiting capacity of cq-PI. Following a one-way analysis of variance, statistical support was provided by a Scheff~ test, which indicated that treatment of cq-PI with 0.19 M of acetaldehyde significantly reduced inhibitory activity (p < 0.05). The pattern is quite similar to the effect of acetaldehyde upon serum. In a study of the effect of acetaldehyde upon the basic pancreatic trypsin inhibitor (Kunitz inhibitor, aprotinin, trasylol), it was noted that the capacity
of aprotinin to inhibit trypsin is not reduced by acetaldehyde. The effect of acetaldehyde upon soybean trypsin inhibitor (SBTI) was also explored. The inhibitory effect of SBTI on trypsin is not influenced upon prior treatment of the SBTI with acetaldehyde.
0.20
e-
E E
_c g: m
o c ¢L 0.10 E g:
0.00
Control
tZl-PI
al--Pf + (o.19 M)
(o.Ts
FIG. 2. Effect of AGSOI-X8 resin-treated 33% acetaldehyde upon the trypsin-inhibiting ability of c~-proteinase inhibitor (mean ± SEM). To 15-#1 aliquots of cq-PI in capped Eppendorf tubes was added 0, 2, or 8/zl of resin-treated 33070acetaldehyde and sufficient 0.2 M Pi (Na+), pH 8.0, to a total volume of 80/~l. The acetaldehyde concentrations were 0.19 M and 0.75 M, respectively. The mixture was preincubated at room temperature for 1 hour, after which time 20/zl of trypsin (20/~g in 1 mM HCI) was added. A trypsin control was also prepared. The mixture was further incubated at room temperature for 30 minutes after which time aliquots of 5/~l were taken for assay at 37* over a l-hour time course, as described in the legend to Fig. 1A (n = 6). (The acetaldehyde concentrations in the incubation mixtures were 1.25 × 10 -4 M and 5 x 10-4 M, respectively.)
DISCUSSION Data have been presented which indicate that acetaldehyde, a product of ethanol metabolism, can lower the trypsininhibiting potential of human serum. Further experiments have shown that the capacity of cq-PI to inhibit trypsin is decreased by prior exposure to acetaldehyde. These observations lead to the suggestion that acetaldehyde might lower cq-PI levels in chronic alcoholics, thereby reducing protection of such individuals from release of accidentally activated trypsinogen into the blood stream. Patients with a higher trypsinlike activity in their blood and with pancreatitis have been reported among alcoholics (15). cq-PI is the major trypsin inhibitor in the blood, although smaller amounts of cq-macroglobulin, cq-antiplasmin, antithromhin III, and inter-c~-inhibitor (a2-MG, c~2-AP, ATIII, and IaI), all of which inhibit trypsin have been reported therein (21). a r P I is active against a number of proteases (21), including elastase, cathepsin G, chymotrypsin, kallikrein, thrombin, plasmin, factor Xa, collagenases, and urokinase. Hence, it is possible to readily envision an extraordinary impact of alcoholism and acetaldehyde reactivity upon a wide spectrum of key proteolytic reactions, including coagulation, fibrinolysis, activation of hormones and zymogens, as well as other intracellular and extracellular processes. The functional groups which are potentially susceptible to reactivity in ~ - P I are found in many cellular and extracellular proteins with defined functions. Aldehydes react covalently with primary amino and primary amide groups (6) as well as guanidyl groups (9-11,18), indole rings (5), imidazole rings (8), and phenolic rings (8). The reaction with primary amines is reversible (18). Reaction of guanidyl groups of arginine with bifunctional aldehydes such as glyoxal leads to a cyclic adduct (11). Crosslinking of functional groups in proteins by formaldehyde to form methylene bridges has also been reported (79). It is reasonable to conclude that reactions of acetaldehyde are comparable with those of formaldehyde and that it can participate in reactions with the susceptible amino acids in proteins (mentioned above). Studies on the key residues involved in the functioning of a~-PI are incomplete. In a recent review by Travis and Salvesen (21), reactions of arg, lys, and tyr have been summarized. It was concluded that alteration of the charge on lys by reaction with maleic anhydride, acetic anhydride, citraconic anhydride, and trinitrobenzene sulfonic acid destroyed inhibitory activity toward trypsin, chymotrypsin and elastase. Alternatively, reductive methylation of lysine residues, which does not affect charge or "bulk" on the lysines, did not affect Ul-PI activity (2,21). Since acetaldehyde and formaldehyde have an analogous effect upon serum antitrypsin activity and cq-PI activity, and would be expected to affect the charge on the lysine residues, it is tempting to consider that lysine residues do play a key role in the activity of cq-PI. The role of arginine in the activity of c~]-PI has not yet been definitively established (21). However, tyr in ~ - P I is modified by tetranitromethane and by acetylimidazole, both of which inactivated antielastase action while not affecting antitrypsin and antichymotrypsin action. Since Fraenkel-Conrat and Olcott have noted crosslinking in proteins by formaldehyde
184
BRECHER A N D PAVLOCK
\ forming methylene bridges involving the active . C - H of the phenolic tyrosine ring (8), an analogous reacUon with acetaldehyde can be predicted. Hence, inactivation of cq-PI by acetaldehyde may well involve covalent interaction with lysine and tyrosine, thereby affecting an elevation of tryptic activity. While tryptophyls have not yet been implicated in a~-PI activity, a reactive tryptophyl has been reported in ATIII, which has 30% analogy to cq-PI (21). Interestingly, acetaldehyde does not appear to inhibit tryptic activity, just
as acetic anhydride does not inhibit the enzyme (5). Similarly, there is no inactivation of the Kunitz inhibitor or SBTI by acetaldehyde. Hence, it appears that chemical modification of proteins may be a possible contributory factor to the generation of an cq-PI deficiency, supplementary to the genetic deficiency (15) with appropriate clinical alterations. Such patterns may be further altered by covalent interactions of aldehydes with RNA and DNA.
REFERENCES 1. Boyer, T. D. In: Wyngaarden, J. B.; Smith, L. H., eds. Cecil textbook of medicine. Philadelphia: Saunders; 1985:835-845. 2. Busby, T. F.; Yu, S. -D.; Gan, J. C. Radioactive labeling of cq-antitrypsin, trypsin, and chymotrypsin by reductive methylation: properties of the labeled derivatives. Arch. Biochem. Biophys. 1984:267-275; 1977. 3. Erlanger, B. F.; Kokowsky, N.; Cohen, W. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95:271-278; 1961. 4. Fraenkel-Conrat, H.; Bean, R. S.; Lineweaver, H. Essential groups for the interaction of ovomucoid (egg white trypsin inhibitor) and trypsin, and for tryptic activity. J. Biol. Chem. 177:395403; 1949. 5. Fraenkel-Conrat, H.; Brandon, B. A.; Olcott, H. S. The reaction of formaldehyde with proteins IV. Participation of indole groups. Gramicidin J. Biol. Chem. 168:99-118; 1947. 6. Fraenkel-Conrat, H.; Cooper, M.; Olcott, H. S. The reaction of formaldehyde with proteins. J. Am. Chem. Soc. 67:950-954; 1945. 7. Fraenkel-Conrat, H.; Mecham, D. K. The reaction of formaldehyde with proteins. VII. Demonstration of intermolecular crosslinking by means of osmotic pressure measurements. J. Biol. Chem. 177:477-486; 1949. 8. Fraenkel-Conrat, H.; Olcott, H. S. Reaction of formaldehyde with proteins. VI. Cross-linking of amino groups with phenol, imidazole, or indole groups. J. Biol. Chem. 174:827-843; 1948. 9. Fraenkel-Conrat, H.; Olcott, H. S. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups. J. Am. Chem. Soc. 70:2673-2684; 1948. 10. Fukui, T.; Kamogawa, A.; Nikuni, Z. Reaction of glyoxal with c~-glycan phosphorylases from potato and rabbit muscle. J. Biochem. (Tokyo) 67:211-218; 1970. 11. Heinrikson, R. L.; Kramer, K. J. Recent advances in the chemical modification and covalent structural analysis of proteins. In:
12. 13.
14.
15. 16.
17. 18. 19. 20. 21.
Kaiser, E. T.; K~zdy, F. J., eds. Progress in bioorganic chemistry. vol. 3. New York: Wiley; 1974:141-249. Keil, B. Reaction of arginine residues in basic pancreatic trypsin inhibitor. FEBS Lett. 14:181-184; 1971. Laskowski, M.; Kato, I. Protein inhibitors of proteinases. In: Snell, E. E.; Boyer, P. D.; Meister, A.; Richardson, C. C., eds. Annual Reviews of Biochemistry. vol. 49. Palo Alto, CA: Annual Reviews Inc.; 1980:593-626. Lehrbach, H.; Diamond, D.; Wozney, J. M.; Boedker, H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 4743-4751; 1977. Levitt, M. D. Pancreatitis. In: Wyngaarden, J. B.; Smith, L. H., eds. Cecil textbook of medicine. Philadelphia: Saunders; 1985: 771-777. McMaster, G. K.; Carmichael, G. G. Analysis of single and double stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74:4835-4848; 1977. Means, G. E.; Feeney, R. E. Reductive alkylation of amino groups in proteins. Biochemistry 7:2192-2201; 1968. Nakaya, K.; Takenaka, O.; Horinishi, H.; Shibata, K. Reactions of glyoxal with nucleic acids, nucleotides, and their component bases. Biochim. Biophys. Acta. 161:23-31; 1968. Nakaya, K.; Horinishi, H.; Shibata, K. States of amino acid residues in proteins. XIV. Glyoxal as a reagent for diserimination of arginine residues. J. Biochem. (Tokyo) 61:345-351; 1967. Shapiro, R.; Hachmann, J. The reaction of guanine derivates with 1,2-dicarbonyl compounds. Biochemistry 5:2799-2807; 1966. Travis, J.; Salvesen, G. S. Human plasma proteinase inhibitors. In: Snell, E. E.; Boyer, P. D.; Meister, A.; Richardson, C. C., Annual Review of Biochemistry. vol. 52. Palo Alto, CA: Annual Reviews, Inc.; 1983:655-709.
0741-8329/92 $5.00 + .00 Copyright©1992PergamonPress Ltd.
Printed in the U.S.A. All rightsreserved.
Acetaldehyde Decreases the Antitryptic Activity of a -Proteinase Inhibitor ARTHUR
S. B R E C H E R 1 A N D J E N N I F E R
L. P A V L O C K
Department o f Chemistry, Bowling Green State University, Bowling Green, OH 43403 Received 20 S e p t e m b e r 1991; A c c e p t e d 6 D e c e m b e r 1991 BRECHER, A. S. AND PAVLOCK J. L. Acetaldehydedecreasesthe antitrypticactivity of arproteinase inhibitor. ALCOHOL 9(3) 181-184, 1992.-The trypsin-inhibiting activity of human serum is lowered upon addition of formaldehyde or acetaldehyde thereto. Acetaldehyde reacts with a-m-proteinase inhibitor (arPI) to decrease its trypsin-inhibiting ability. Acetaldehyde has only a slight effect on the tryptic hydrolysis of benzoyi-DL-arginine-p-nitroanilide. It did not decrease the inhibitory activity of the Kunitz inhibitor (Aprotinin) or soybean trypsin inhibitor. Since aldehydes form covalent products with primary amines, primary amides, arginine, tyrosine, and tryptophan in protein, as well as methylene bridges thereby crosslinking functional groups, it is proposed that one or more such interactions affect at-PI activity. It is further suggested that chronically high levels of acetaldehyde, as a metabolic product of ethanol, may be a contributory factor to the generation of pancreatitis in alcoholics by possibly lowering the effective cq-PI level which is a natural protective element from proteolysis by trypsin. Trypsin
aj-Proteinase inhibitor
Acetaldehyde
Human serum
Alcohol
Alcoholism
Pancreatitis
In view of the observation of pancreatitis in some alcoholics afflicted with cirrhosis, and in view of the natural metabolic transformation of ethanol to acetaldehyde, it was hypothesized that acetaldehyde may react with naturally occurring trypsin inhibitors in liver and serum, thereby causing physiological inactivation of the inhibitors. It was further hypothesized that trypsin-like activity would be elevated as a result of lowered antitrypsin activity, thereby promoting the development of pancreatitis. This communication reports on the lowering of trypsin-inhibiting activity of human serum upon treatment of the serum with acetaldehyde or formaldehyde, as well as the reduction of the trypsin-inhibiting activity of a~-proteinase inhibitor (,~rPI) upon preincubation with acetaldehyde, supporting the suggestion that chemical modification of trypsin inhibitors, in addition to genetic factors (15) and impairment of protein synthesis (1), might influence the onset of pancreatitis.
ACUTE pancreatitis is a condition which is characterized, in part, by an increase in pancreatic enzymes in the serum. Among the enzymes observed to be elevated are amylase, lipase, and a trypsin-like activity (15). As a result of activation of trypsin and other proteases, digestion of the pancreas and peripancreatic tissues is observed. One of the principle causes of pancreatitis in the Western world is the consumption of alcoholic beverages, although numerous other causes have been implicated (15). Alcohol uptake by humans is a major factor in the generation of cirrhosis of the fiver (1). Pediatric arantitrypsin deficiency may also induce cirrhosis of the liver (1). Ethanol uptake causes morphological and functional changes in the hepatocytes. It is metabolized to acetaldehyde which may react with cellular components in the hepatocyte and elsewhere since it circulates freely (1). Aldehydes are highly reactive. They are reported to react with proteins (10,17), thereby affecting conformation and function. Aldehydes are utilized to denature nucleic acids by reaction with amino functional groups and ring nitrogen (14,16,18), as observed with adenine, guanine, uracil, and cytosine in nucleic acids (18,20). Various amino acids in proteins also form covalent products with aldehydes. Primary amines, primary amides, guanidyl groups, indole groups, and phenolic groups are susceptible to modification by formaldehyde leading in some cases to crosslinking (5-9). Modification of ribonuclease A by formaldehyde results in a 40% reduction in enzymic activity within 2 minutes (17), and further reduction in activity with longer exposure of the enzyme to the aldehyde.
EXPERIMENTAL Trypsin, 2 x crystalline, was purchased from Nutritional Biochemical Corp. (Cleveland, OH). Benzoyl-DL-arginine-pnitroanilide (BAPA) and soybean trypsin inhibitor (SBTI) were obtained from Mann Research Laboratories (New York, NY). t~rPI and aprotinin, the basic Kunitz inhibitor, were purchased from Sigma Chemical Co. (St. Louis, MO). AG501-X8 mixed bed resin was supplied by Bio-Rad (Richmond, CA).
J To whom reprint requests should be addressed. 181
182
BRECHER AND PAVLOCK
o.41
(A)
(B)
@.6
o.E =-
'E 0.4
e-
==
~ 0.2
O L..
=. G-J
,~
&
R
/ 0.1
0.0
Control
Serum
.~rum + f (0.29 M)
0.0
~ (1.16 M)
Control
Serum
Serum ÷ (0.19 M) (0.75 M)
(1.5 M)
FIG. 1. Effect of formaldehyde on the trypsin-inhibiting ability of human serum (mean + SEM). (A) To 25-/zl aliquots of human serum in capped Eppendorf tubes were added 0, 2, and 8/zl of 37% formalin and sufficient 0.2 M Pi (Na÷), pH 8.0, to give of total volume of 80/xl. The formaldehyde concentrations were 0.29 and 1.16 M, respectively. The mixture was preincubated at room temperature for 1 hour, after which time 20 t~l of trypsin (20/xg in 1 mM HC1) were added. A control trypsin without serum or formaldehyde was also prepared. After further incubation at room temperature for 30 minutes, aliquots of 10/zl were added to 5 ml of buffered substrate and 990/A of dH20. The solutions were incubated at 370C for 30 minutes, and the reaction was stopped by the addition of 1 ml of 30% acetic acid. The release of p-nitroaniline was quantitated by measuring A4~0(n = 4). (The formaldehyde concentrations in the incubation mixtures were 3.8 x 10 -4 M and 1.5 x 10 -3 M, respectively.) (B) Effect of acetaldehyde upon the trypsin-inhibiting ability of human serum (mean + SEM). To 25-/A aliquots of human serum in capped Eppendorf tubes were added 2, 8, and 16/zl of resin-treated 33% acetaldehyde and sufficient 0.2 M Pi (Na+), pH 8.0, to a total volume of 80/~1. The acetaldehyde concentrations were 0.19 M, 0.75 M, and 1.5 M, respectively. The mixture was preincubated at room temperature for 1 hour, after which time 20 #1 of trypsin (20/zg in 1 mM HC1) were added. A trypsin control was also prepared. After standing an additional 30 minutes at room temperature, 10/zl of the mixtures were taken for assay as described for (A) (n = 4). (The acetaldehyde concentrations in the incubation mixtures were 2.5 × 10 -4 M, 1 × 10 -4 M, and 2 × I0 -3 M, respectively.)
Trypsin activity was quantitated by the procedure o f Erlanger et al. (3) which measures the release o f p-nitroaniline from benzoyl-DL-arginine-p-nitroaniline'HC1 (BAPA). B A P A (43.5 mg) was taken up in 1 ml dimethylsulfoxide, and the solution was diluted to 100 ml with 0.05 M Tris/HC1, p H 8.2/0.02 M CaC12. To 5 ml o f buffered substrate was added 20/~l trypsin (1 m g / m l in 1 m M HCI) and sufficient distilled H20 to a total o f 6 ml. The solution was incubated in a shaking water bath at 37°C for 30 or 60 rain, as indicated in the Fig. 1 legend, after which time the reaction was terminated by the addition o f 1 ml of 30% acetic acid. The p-nitroaniline generated during the course of the reaction was determined by reading the absorption at 410 nm. Conditions for studying the effect of serum, formaldehyde, acetaldehyde, and tx~-PI on tryptic activity are given in the Figure legends. Aliquots o f formaldehyde and acetaldehyde were passed through short
columns of AG-501-X8 mixed bed resin in order to remove oxidation products (formic and acetic acids). (It was observed, however, that formaldehyde and acetaldehyde exhibited the same effect on the proteins with or without exposure to the resin.) Acetaldehyde gave a negative response for the presence of peroxides with the potassium idodide reaction and upon reaction with ferrous a m m o n i u m sulfate and potassium thiocyanate. RESULTS The data in Fig. 1 reflect the effect of aldehydes upon the ability o f human serum to inhibit the tryptic hydrolysis of B A P A . Figure 1A indicates the effect o f preincubation of serum at r o o m temperature with formaldehyde upon its capacity to inhibit tryptic activity. Tryptic activity in formalde-
ALDEHYDES INACTIVE aI-PROTEINASE INHIBITOR
183
hyde-treated serum is essentially tripled, as compared with its activity in an untreated serum mixture. Results are comparable when formaldehyde is passed through a Bio-Rad AG-501X8 mixed bed resin to remove oxidation products (data not shown). Figure IB shows that treatment of serum with resintreated acetaldehyde similarly decreases the trypsin-inhibiting capacity of the serum. Acetaldehyde, of itself, has only a very slight effect upon tryptic activity. Since cq-PI is the major trypsin inhibitor in serum, the effect of acetaldehyde upon its physiological effect was examined. The data in Fig. 2 indicate that pretreatment of cq-PI with acetaldehyde considerably reduces the trypsin-inhibiting capacity of cq-PI. Following a one-way analysis of variance, statistical support was provided by a Scheff~ test, which indicated that treatment of cq-PI with 0.19 M of acetaldehyde significantly reduced inhibitory activity (p < 0.05). The pattern is quite similar to the effect of acetaldehyde upon serum. In a study of the effect of acetaldehyde upon the basic pancreatic trypsin inhibitor (Kunitz inhibitor, aprotinin, trasylol), it was noted that the capacity
of aprotinin to inhibit trypsin is not reduced by acetaldehyde. The effect of acetaldehyde upon soybean trypsin inhibitor (SBTI) was also explored. The inhibitory effect of SBTI on trypsin is not influenced upon prior treatment of the SBTI with acetaldehyde.
0.20
e-
E E
_c g: m
o c ¢L 0.10 E g:
0.00
Control
tZl-PI
al--Pf + (o.19 M)
(o.Ts
FIG. 2. Effect of AGSOI-X8 resin-treated 33% acetaldehyde upon the trypsin-inhibiting ability of c~-proteinase inhibitor (mean ± SEM). To 15-#1 aliquots of cq-PI in capped Eppendorf tubes was added 0, 2, or 8/zl of resin-treated 33070acetaldehyde and sufficient 0.2 M Pi (Na+), pH 8.0, to a total volume of 80/~l. The acetaldehyde concentrations were 0.19 M and 0.75 M, respectively. The mixture was preincubated at room temperature for 1 hour, after which time 20/zl of trypsin (20/~g in 1 mM HCI) was added. A trypsin control was also prepared. The mixture was further incubated at room temperature for 30 minutes after which time aliquots of 5/~l were taken for assay at 37* over a l-hour time course, as described in the legend to Fig. 1A (n = 6). (The acetaldehyde concentrations in the incubation mixtures were 1.25 × 10 -4 M and 5 x 10-4 M, respectively.)
DISCUSSION Data have been presented which indicate that acetaldehyde, a product of ethanol metabolism, can lower the trypsininhibiting potential of human serum. Further experiments have shown that the capacity of cq-PI to inhibit trypsin is decreased by prior exposure to acetaldehyde. These observations lead to the suggestion that acetaldehyde might lower cq-PI levels in chronic alcoholics, thereby reducing protection of such individuals from release of accidentally activated trypsinogen into the blood stream. Patients with a higher trypsinlike activity in their blood and with pancreatitis have been reported among alcoholics (15). cq-PI is the major trypsin inhibitor in the blood, although smaller amounts of cq-macroglobulin, cq-antiplasmin, antithromhin III, and inter-c~-inhibitor (a2-MG, c~2-AP, ATIII, and IaI), all of which inhibit trypsin have been reported therein (21). a r P I is active against a number of proteases (21), including elastase, cathepsin G, chymotrypsin, kallikrein, thrombin, plasmin, factor Xa, collagenases, and urokinase. Hence, it is possible to readily envision an extraordinary impact of alcoholism and acetaldehyde reactivity upon a wide spectrum of key proteolytic reactions, including coagulation, fibrinolysis, activation of hormones and zymogens, as well as other intracellular and extracellular processes. The functional groups which are potentially susceptible to reactivity in ~ - P I are found in many cellular and extracellular proteins with defined functions. Aldehydes react covalently with primary amino and primary amide groups (6) as well as guanidyl groups (9-11,18), indole rings (5), imidazole rings (8), and phenolic rings (8). The reaction with primary amines is reversible (18). Reaction of guanidyl groups of arginine with bifunctional aldehydes such as glyoxal leads to a cyclic adduct (11). Crosslinking of functional groups in proteins by formaldehyde to form methylene bridges has also been reported (79). It is reasonable to conclude that reactions of acetaldehyde are comparable with those of formaldehyde and that it can participate in reactions with the susceptible amino acids in proteins (mentioned above). Studies on the key residues involved in the functioning of a~-PI are incomplete. In a recent review by Travis and Salvesen (21), reactions of arg, lys, and tyr have been summarized. It was concluded that alteration of the charge on lys by reaction with maleic anhydride, acetic anhydride, citraconic anhydride, and trinitrobenzene sulfonic acid destroyed inhibitory activity toward trypsin, chymotrypsin and elastase. Alternatively, reductive methylation of lysine residues, which does not affect charge or "bulk" on the lysines, did not affect Ul-PI activity (2,21). Since acetaldehyde and formaldehyde have an analogous effect upon serum antitrypsin activity and cq-PI activity, and would be expected to affect the charge on the lysine residues, it is tempting to consider that lysine residues do play a key role in the activity of cq-PI. The role of arginine in the activity of c~]-PI has not yet been definitively established (21). However, tyr in ~ - P I is modified by tetranitromethane and by acetylimidazole, both of which inactivated antielastase action while not affecting antitrypsin and antichymotrypsin action. Since Fraenkel-Conrat and Olcott have noted crosslinking in proteins by formaldehyde
184
BRECHER A N D PAVLOCK
\ forming methylene bridges involving the active . C - H of the phenolic tyrosine ring (8), an analogous reacUon with acetaldehyde can be predicted. Hence, inactivation of cq-PI by acetaldehyde may well involve covalent interaction with lysine and tyrosine, thereby affecting an elevation of tryptic activity. While tryptophyls have not yet been implicated in a~-PI activity, a reactive tryptophyl has been reported in ATIII, which has 30% analogy to cq-PI (21). Interestingly, acetaldehyde does not appear to inhibit tryptic activity, just
as acetic anhydride does not inhibit the enzyme (5). Similarly, there is no inactivation of the Kunitz inhibitor or SBTI by acetaldehyde. Hence, it appears that chemical modification of proteins may be a possible contributory factor to the generation of an cq-PI deficiency, supplementary to the genetic deficiency (15) with appropriate clinical alterations. Such patterns may be further altered by covalent interactions of aldehydes with RNA and DNA.
REFERENCES 1. Boyer, T. D. In: Wyngaarden, J. B.; Smith, L. H., eds. Cecil textbook of medicine. Philadelphia: Saunders; 1985:835-845. 2. Busby, T. F.; Yu, S. -D.; Gan, J. C. Radioactive labeling of cq-antitrypsin, trypsin, and chymotrypsin by reductive methylation: properties of the labeled derivatives. Arch. Biochem. Biophys. 1984:267-275; 1977. 3. Erlanger, B. F.; Kokowsky, N.; Cohen, W. The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95:271-278; 1961. 4. Fraenkel-Conrat, H.; Bean, R. S.; Lineweaver, H. Essential groups for the interaction of ovomucoid (egg white trypsin inhibitor) and trypsin, and for tryptic activity. J. Biol. Chem. 177:395403; 1949. 5. Fraenkel-Conrat, H.; Brandon, B. A.; Olcott, H. S. The reaction of formaldehyde with proteins IV. Participation of indole groups. Gramicidin J. Biol. Chem. 168:99-118; 1947. 6. Fraenkel-Conrat, H.; Cooper, M.; Olcott, H. S. The reaction of formaldehyde with proteins. J. Am. Chem. Soc. 67:950-954; 1945. 7. Fraenkel-Conrat, H.; Mecham, D. K. The reaction of formaldehyde with proteins. VII. Demonstration of intermolecular crosslinking by means of osmotic pressure measurements. J. Biol. Chem. 177:477-486; 1949. 8. Fraenkel-Conrat, H.; Olcott, H. S. Reaction of formaldehyde with proteins. VI. Cross-linking of amino groups with phenol, imidazole, or indole groups. J. Biol. Chem. 174:827-843; 1948. 9. Fraenkel-Conrat, H.; Olcott, H. S. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups. J. Am. Chem. Soc. 70:2673-2684; 1948. 10. Fukui, T.; Kamogawa, A.; Nikuni, Z. Reaction of glyoxal with c~-glycan phosphorylases from potato and rabbit muscle. J. Biochem. (Tokyo) 67:211-218; 1970. 11. Heinrikson, R. L.; Kramer, K. J. Recent advances in the chemical modification and covalent structural analysis of proteins. In:
12. 13.
14.
15. 16.
17. 18. 19. 20. 21.
Kaiser, E. T.; K~zdy, F. J., eds. Progress in bioorganic chemistry. vol. 3. New York: Wiley; 1974:141-249. Keil, B. Reaction of arginine residues in basic pancreatic trypsin inhibitor. FEBS Lett. 14:181-184; 1971. Laskowski, M.; Kato, I. Protein inhibitors of proteinases. In: Snell, E. E.; Boyer, P. D.; Meister, A.; Richardson, C. C., eds. Annual Reviews of Biochemistry. vol. 49. Palo Alto, CA: Annual Reviews Inc.; 1980:593-626. Lehrbach, H.; Diamond, D.; Wozney, J. M.; Boedker, H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 4743-4751; 1977. Levitt, M. D. Pancreatitis. In: Wyngaarden, J. B.; Smith, L. H., eds. Cecil textbook of medicine. Philadelphia: Saunders; 1985: 771-777. McMaster, G. K.; Carmichael, G. G. Analysis of single and double stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74:4835-4848; 1977. Means, G. E.; Feeney, R. E. Reductive alkylation of amino groups in proteins. Biochemistry 7:2192-2201; 1968. Nakaya, K.; Takenaka, O.; Horinishi, H.; Shibata, K. Reactions of glyoxal with nucleic acids, nucleotides, and their component bases. Biochim. Biophys. Acta. 161:23-31; 1968. Nakaya, K.; Horinishi, H.; Shibata, K. States of amino acid residues in proteins. XIV. Glyoxal as a reagent for diserimination of arginine residues. J. Biochem. (Tokyo) 61:345-351; 1967. Shapiro, R.; Hachmann, J. The reaction of guanine derivates with 1,2-dicarbonyl compounds. Biochemistry 5:2799-2807; 1966. Travis, J.; Salvesen, G. S. Human plasma proteinase inhibitors. In: Snell, E. E.; Boyer, P. D.; Meister, A.; Richardson, C. C., Annual Review of Biochemistry. vol. 52. Palo Alto, CA: Annual Reviews, Inc.; 1983:655-709.
