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CLIN. CHEM. 29/3, 447-451 (1983) Kinetic Assay of Human Pepsin with Albumin-Bromphenol Substrate Blue as Stephen P. Gray and John A. Billings A novelsubstrate,albumincomplexedwith bromphenolblue, has been developed for the assay of human gastric juice pepsin by a kinetic method in the Cobas centrifugal analyzer. The action of pepsin on the complex degrades the albumin and releases the dye. The change in the color of the substrate is a zero-order reaction. Human and porcine pepsin have different Km’Swith the new substrate. This kinetic methodhas a throughputof 28 tests in approximately10 mm and good precision (CV = 2.0%). Other advantages are analysis in homogeneous solution (thereby eliminating the need to separate substrate and products), lack of interfer- ence from bilirubin or phenol red, and the expression of pepsin activity in IUB enzyme units. AdditionalKeyphrases:gastric juice sis centrifugal analydifferences between human and porcine pepsin Pepsins (EC 3.4.23.1) are secreted in gastric juice as inactive pepsinogens, of which eight are known (1). The pepsinogens are cleaved at pH 5 to form the actiye enzyme. Once cleavage has begun, it proceeds autocatalytically in the hydrochloric acid normally present in the stomach. The main source of pepsin is the chief cell located in small pits in the gastric mucosa. In the stomach, pepsins appear to act primarily on peptide bonds in the middle of the protein molecule, which are made accessible by hydrochloric acid denaturation; thus, the active pepsins are endopeptidases and show the greatest activity against aromatic amino acids such as tryptophan, phenylalanine, and tyrosine (2). The assay of pepsin depends on estimating the number of peptide bonds broken during the rcaction with a protein substrate. Some authors have used dried plasma proteins (3) or edestin (4) as a substrate, but bovine hemoglobin and albumin have been the most widely used. In general the substrate is incubated at pH 2.0 with the pepsin (optimal pH range 1.8-3.5) and the liberated tyrosine is then reacted with Folin phenol reagent (5) or the absorbance is read at 275-280 tim (6). Most such methods compare the enzyme activity vs that of a reference preparation of hog mucosal pepsin, which differs in potency according to the degree of purification, and the units of pepsin activity are then reported in terms of “tyrosine equivalents,” “hog pepsin units.” or “absorbance units” (6). Use of protein substrates suffers from the technical drawback that the released soluble products of pepsin digestion have the same optical and chemical properties as the intact amino acids in the protein chain and therefore have to be physically separated from the original substrate, usually by centrifugation, ifitration, or dialysis, before the change in absorbance can be measured. Such procedures militate against the development of kinetic methods of analysis for the enzyme, which require homogeneous solution chemistry. Furthermore, the different expressions for the units of enzyme activity prevent accurate comparisons with results from other investigative centers. In general, research groups have tended to establish their own reference ranges for the given experiment, with the result that the reported values often do not have universal applicability. Moreover, no pure, stable human pepsin is yet available as a reference standard. Most enzymes are measured kinetically in clinical chemical laboratories, which also takes advantage of the various automated analyzers devised specifically for that purpose. The need for a new method of measuring human gastric pepsin by reaction-rate analysis in homogeneous solution, such that the activity could be expressed in standardized units in common with most other enzymes, is undeniable. The present study attempts to fulfill it. Materials and Methods Human gastric juice pepsin having its optimal activity around pH 2.0 (2), we decided to measure at that pH, using glycine HC1 buffer (0.4 mol/L) to maintain it. Buffer. Glycine HC1, 0.4 molJL, pH 2.0 at 37 “C; reagentgrade glycine was obtained from BDH, Poole, Dorset, U.K. Albumin. Bovine albumin was purchased as “Albumin Stock Solution,” 10 g/100 mL, code no. 905-10 (Sigma Chemical Co., St. Louis, MO 63178). Bromphenol blue. Purchased as a thy powder, “pH Indicator” (Hopkin and Williams, Chadwell Heath, Essex, U.K.). Dissolve the appropriate amount of dye (Mr 670.02) in the minimum amount of ethanol, dilute to the mark with pH 2.0 glycine buffer, and recheck the pH. The stock solution was 1.0mmol/L. Substrate. For use in the automatic analyzer, add 1.80 mL of the Albumin Stock Solution to 3.0 mL of the bromphenol blue stock solution, mix well, and make up to 20 mL with pH 2.0 glycine buffer; re-mix. This reagent can be placed in the reagent tray ready for use. Its final concentration in the cuvette is 150 prnol/L. Samples. Gastric juice, ifitered or centrifuged before analysis. Procedures Substrate development. Albumin combines with bromphenol blue at pH 2.0 to give a product having a different color from the original dye. Pepsin acting on this complex appears to break it up and regenerate the free bromphenol blue. These color changes can be followed over time. Composition of the albumin-bromphenol blue complex. We determined the composition of the complex by application of the law of mass action to the assumed equilibrium: k of Biochemistry, Royal Naval Hospital, Gosport, Hampshire P012 2AA, U.K. Received Oct. 15, 1982; accepted Dec. 14, 1982. Department Haslar, niALB + nBPB where k is the stability ALBm constant, - BPB ALI3 represents albumin, CLINICALCHEMISTRY,Vol.29, No. 3, 1983 447 BPB is bromphenol blue, and m and n are the numbers molecules reacting. Rearranging, k = of raphy on activated [ALBmBPBnI [ALB]m- [BPB]” from the double-log plot provide an estimate of the values fcr m and n, thereby giving the molar composition of the complex. To calculate the stability constant, k, we substituted appropriate pairs of related data into the rearranged expression shown above. Spectrophotometry. The spectral chatiges of the new complex with time in the presence of human gastric juice pepsin were examined with a Cary 219 recording spectrophotometsr, in an attempt to determine the optimum wavelength for studying the reaction. Kinetics. Curves relating absorbance change with the new substrate to differing pepsin concentrations were plotted to calculate Km and V, and hence the optimal substrate concentration. Conditions for zero-order rate reaction were established by studying the time course of the pepsinsubstrate reaction. Km and V, were calculated by using the Hanes plot (7). We also examined the effect of temperature on the reaction and constructed an Arrhenius plot of the data relating temperature to pepsin activity, from which we calculated the activation energy and the temperature coefficient, Qio, the factor by which the rate increases when the temperature is increased by 10#{176}C. Auto,nated analysis. We adapted the manual spectrophotometer assay for pepsin to the Cobas Bio Centrifugal Analyzer (Roche Ltd., Welwyn Garden City, U.K.) because we found that the period of zero-order kinetics was difficult to determine manually. The Cobas, however, had the facility to detect the linear portion of the reaction and then calculate the best fit by a least-squares regression analysis. Use of this instrument allowed us to express the results in terms of IUB enzyme units (U) of activity at 37#{176}C and to automate the whole assay for pepsin. Correlation with a reference method of pepsin assay. We used the method of Burstad (6), which correlates very closely with the earlier, classical method of Anson (5). The latter is used in the United States, whereas the Burstad method is popular in Europe. We analyzed 92 samples of human gastric juice obtained post-stimulation with pentagastrin and insulin and calculated the correlation. The Burstad method in outline is as follows. Add diluted gastric juice to human hemoglobin substrate in HC1 at pH 2.0 and incubate at 25 #{176}C for 10 min. Then, to the mixture add trichloroacetic acid to precipitate the unchanged substrate proteins. Filter these off and read the absorbance of the ifitrate at 280 rim, expressing the pepsin activity in absorbance units, or compare the results with those for a reference porcine pepsin preparation treated in the same way as the gastric juice samples. Action of pepsin on the new substrate. The action of pepsin on albumin is well known (2), but we had to test the possibility that complexation with bromphenol blue may alter the chemistry of the reaction. To test this thesis, porcine pepsin (Boehringer Mannheim, BCL, Lewes, Sussex, U.K.) and human gastric pepsin were allowed to react 448 CLINICALCHEMISTRY,Vol.29, No.3, 1983 silica gel (8). between IUB units and Anson units. Although many pepsin units are in current use, we thought we Relationship We set up two experiments, in one keeping the albumin concentration constant and varying that of the bromphenol blue, the second keeping the bromphenol blue constant and varying the albumin. The absorbances of all the solutions were measured at 605 nm and the values of m and n were evaluated by plotting the logarithms of absorbances against the logarithms of the molar concentrations of each reactant. The slopes of the curves with albumin alone and with albumin-bromphenol blue at pH 2.0 in 0.4 mol/L glycine HC1 buffer until apparent equilibrium was attained. The products of digestion were then analyzed for free amino acids by thin-layer chromatog- could establish the relationship between IUB enzyme units (1 U = 1 mol of substrate converted per minute) and Anson units (5), in which the activity of commercial porcine preparations is commonly expressed. One Anson unit is the enzyme activity that liberates under specified assay conditions sufficient tyrosine to increase the absorbance at 280 rim by 0.001/ruin. We tested known amounts of porcine pepsin with the new assay system and calculated the equivalent in IUB enzyme units. Possible interferences. Bilirubin, not normally present in gastric juice, may occasionally appear as a consequence of duodenal refiux. To check whether it interfered with the new assay, we obtained specimens of gastric juice heavily contaminated with bilirubin from such reflux, mixed them with samples of clear, uncontaminated gastric juice, and reanalyzed the resulting mixtures. Phenol red, a “nonabsorbable ion” given by constant infusion via the stomach tube to provide an estimate-by calculating its recovery-of the extent of gastric gains and losses by reflux, was tested as a possible interferent by addition in various proportions (from zero to 100% dye) to the gastric juice and re-analyzing. Statistical methods. Linear-regression analysis, mean and SD, and Student’s t-test were taken from Colquhoun (9). For all calculations we used an HP-9815 desk-top calculator and the statistical programs provided by the manufacturer (Hewlett Packard Ltd., Wokingham, U.K.). Protocol for pepsin assay on the Cobas Bio. In principle, substrate is pipeted by the instrument into the rotor cuvette equilibrated to 37 “C; gastric juice (10-20 tL) is then added and mixed. The first reading is taken 0.5 s after this mixing and sibsequent readings made at 10-s intervals for 5 min. The Cobas measures the absorbance change per minute, searches for the start of a linear path of decreasing absorbance at 605 nm, and calculates a least-squares regression analysis on the maximum number of points lying within a bandwidth of ±0.0025 absorbance units (A). A typical format for the analysis is as follows: 1. Units U/L 2. Calculation factor 1041 (incorporates molar absorptivity) 3. Temperature, #{176}C 37.0 4. Type of analysis 5. Wavelength, 6. Sample volume, 7. Diluent volume, 8. Reagent volume, 9. Incubation time, s Time of first read- 2 tim (kinetic, decreasing sorbance) ab- 605 10. ing, 11. 12. 13. S Time interval,s No. of readings 20 (gastric juice) 20 (distilled water) 200 (substrate) 180 (before sample addition) 0.5 10 30 1 (blanks after all added to cuvette) The centrifugal rotor holds 28 samples of gastric juice, which are analyzed concurrently. The incubation interval before analysis is 3 mm and the analysis time is 5 mm; Blanking mode I.--. I 04 . -----4------ -0.-------- 04 05 - DI 10gb / - - - I 1* 4 i. 1I UiS, hauL n,JI....l Fig. 1. Logarithmic plot to determine binding parameters for the albumin-bromphenol blue reaction The slope of this double-icy plot equates to the numericalvalue of the molar ratios,m and n, inthe equationfor k (see text for details) therefore, the time taken for the 28 tests to be completed is about 10 mm. Results Composition of the albumin-bromphenol blue complex. Using the logarithmic method described in Methods, we determined that albumin and bromphenol blue react in a one-to-one molar ratio. The value of k, the stability constant, was 3.72 x i0, which indicates that very little free dye or albumin is present when the two compounds are allowed to react in unimolar proportions. One of the curves is illustrated in Figure 1. Spectral properties of the complex. In Figure 2 the spectral scans of the free bromphenol blue and the complex with albumin are illustrated superimposed to show the change in absorption. There are two absorption peaks, at 450 nm and 605 tim; we chose to monitor the one at 605 nm for the reaction with pepsin because the absorbance values there are much lower and thus in the range of most spectrophotometers. Using this wavelength meant that a decrease in absorbance with time was monitored. When the enzyme reaction was allowed to go to equilibrium, the spectral absorbance of the products was indistinguishable from that of the uncomplexed dye. Kinetics of the reaction. The rate of change of absorbance per minute was plotted against a series of different substrate concentrations reacted with a high activity sample of human gastric juice. The resulting data wlore plotted according to the Hanes procedure (7). The results of 12.0 prnolJL for :Ji .-“ I, /. F I / / r ,/ / ‘ \ / I I., I I I , s___, 0.50 ‘ 00 400 450 500 560 600 ,eo nm Fig. 2. The change in absorbance at 605 nm when the substrate, albumin-bromphenol blue (curve 2), is aftacked by pepsin at pH 2.0 to yield the free bromphenol blue (curve 1) p710 -Km 0 (ANumIn-Brompheeol Blus)[S),p mol/L Fig. 3. Effect of varying the substrate(albumin-bromphenolblue) concentration [S] in the presence of a constant amount of human pepn y.axis, LSVA substrateooncentration/absorbance = and of 0.341 mol/min for V were checked by means of an iterative calculation for each (7) and both techniques were in agreement. An illustrative Hanes plot is shown in Figure 3. To ensure saturation of the enzyme, we used a final substrate concentration of approximately 10-fold the Km value, which gave a zero-order rate reaction. The absorbance value of this concentration of substrate was approximately 1.2 A, which is well in the range of most enzyme analyzers. The molar absorptivity for the complex was 12 x io L mol cm1 at 605 rim. Data obtained with the Cobas analyzer, which can read the absorbance every 10 s until the time to equilibrium, indicated that the time needed to reach zero-order rate was a function of the pepsin concentration. It was necessary to follow each test for 5 rain to accumulate sufficient data points for a least-squares regression analysis within the close limits set by the Cobas analyzer. Specimens of human gastric juice with low pepsin activity showed a “lag” phase of up to 1 mm before the linear path commenced; in samples of high activity this lag phase lasted only a few seconds. Effect of temperature on the reaction. We used a specimen of human gastric juice with high peptic activity to study the effect of increasing temperature on the rate of the reaction. The plot of the data is shown in Figure 4 as an Arrhenius diagram. The calculated activation energy was 74.88 kJ/ mol, and the temperature coefficient, Qio, was 2.5, indicating that the rate of the reaction was multiplied by that amount for each 10#{176}C rise in temperature. There was no evidence of a transition point in the range of temperatures studied. Correlation with a comparison method. We used the method of Burstad (6) as a reference for pepsin assay. Samples of gastric juice aspirated after stimulation with pentagastrin and insulin were assayed for pepsin activity by the new method and by the method of Burstad. The resultKm - CLINICALCHEMISTRY, Vol. 29, No. 3, 1983 449 4. 50 44 42 4 3 6 Activation Qio ‘. - - - - - - - e#{176}C pepsin had an activity 4 74 Energy kJ/mol 2.5. - - - - - - - - - -. - “c;; - R20.982 - - - - - - N 4.50 - 4.00 - 310 - - 312 - - 314 - - 3* - 315 - - - - 320 - - - - - - 5 324 - - 330 5 2 1/OK (x105) Fig. 4. Arrhenius plot of the effect of changing the temperatureat which human pepsin reacts with albumin-bromphenol blue The high value for Q,, indicatesthat the enzymes specific activity increases rapidly with increasing temperature of 2500 Anson units (5) per milligram; therefore, 16 pg would have a specific activity of approximately 40 Anson units. Thus the approximate relationship between units was: 1 Anson unit 10 UIL. Kinetics of porcine pepsin. The same porcine pepsin was investigated for its kinetic properties in the new substrate. We found that its Km was 80 prnol/L, i.e., six- to sevenfold the value for human pepsin found in the gastric juice from healthy people. Within-run analysis. We re-analyzed gastric juice samples and subjected the data to linear-regression analysis and the paired t-test. In a typical set of results (n = 15) we found the following: means 124.01 and 124.11 UIL and the t-test for paired values 0.1 (CV = 2.0%). The range of values tested was 20.29-387.28 UIL (SD 94.8 UIL). Between-run analysis. We carried out these analyses on fresh samples. The CV was 1.86%, and the mean differences were statistically insignificant. Stability studies. We investigated the effect on pepsin stability of storage in the frozen state. We analyzed 20 samples with values ranging from 3.3 to 181.0 U/L before and after storage at -20 #{176}C. The mean value before storage was 95.6 UIL and after 93.8 UIL. The t-value was significant at the 5% level and we concluded there was a change in pepsin recovery after freezing and storage at this temperature. On the other hand, stability studies on pepsin in gastric juice stored at -85 #{176}C indicated no significant deterioration at that temperature. Interference from additional substances. The commonest contaminant of gastric juice is bile derived from duodenal refiux, so we added duodenal juice to gastric juice and reassayed the samples for pepsin by the new method. We analyzed 20 samples and found no significant difference between the original values and those after addition of bile (t 240am Fig. 5. Correlation between the new automated kinetic method with albumin-brornphenol blue substrate (y) and the manual method of Burstad (6) (Jc) Thesetwo very differentmethodsagreeclosely( 0.984) over a wide range of enzymeactivities.The regressionequationis: pepsin, U/L -4.41 + 251.3 A (at 280 nm). SD of the x-varlable 0.43 (mean 0.62) and SD of the y.varlable = 108.0(mean 151.6) = = ing data for 92 specimens are illustrated in Figure 5. The correlation was good, r2 = 0.984. Both methods gave identical post-stimulation patterns and appear to-be measuring the same thing. Effect of pepsin on the substrate. To check that complexing bromphenol blue with albumin did not alter fundamentally the chemistry of the action of pepsin on the albumin part of the complex, we used thin-layer chromatography in butanoll acetic acid/water (60/30/10, by vol) to separate the products after digestion. The amino acid pattern from the albumindye complex was indistinguishable from that for uncomplexed albumin. We concluded, therefore, that not only did the dye not materially affect the protein digestion by pepsin but also the results were compatible with the idea that the enzyme attacked the albumin to release amino acids and detached the dye from the remnant protein. Correlation between enzyme units and Anson units.”Porcine pepsin, prepared from crystalline pepsin and lyophilized, was studied in the new assay system in the same way as human gastric juice pepsin. Analysis of 10 samples of 20 1L, each containing 16 &g of porcine pepsin, gave the following results: mean 418.5 UIL (at 37#{176}C), SD 8.6 U/L, and CV 2.0%. According to the manufacturer, the porcine 450 CLINICAL CHEMISTRY, Vol. 29, No. 3, 1983 = 0.53). Another contaminant of gastric juice in experimental work is phenol red, used to determine reflux losses, and we used a similar procedure to the above, adding the dye in several proportions to the gastric juice. The mean value before addition was 137.0 UIL and after the mean was 138.6 UIL (t = 0.92). We concluded that phenol red does not interfere with the assay. Normal values. The normal range for pepsin cannot be established in the usual way because the enzyme is secreted in response to many physiological and psychological factors. We report basal (i.e., nonstiniulated) values for pepsin, which we found to be about 2.5-10.2 U/h (mean ±2 SD). Discussion The chemistry of the reaction of albumin-bromphenol blue with pepsin is not simple. Albumin combines with the dye at pH 2.0 to give a dark red compound that has a slight green fluorescence. During attack by the enzyme the color changes to give the yellow green of the free bromphenol blue and the fluorescence disappears. The complexed dye does not appear to alter the types or amounts of amino acids released from albumin by digestion with the pepsin, so we assume that the protein part of the substrate is degraded in the usual manner (2) and that the previously bound dye is then detached from the digested remnants. If the rate of release of the dye is a function of the enzyme activity, as it appears to be, then we can use it as an assay for pepsin. By regulating the conditions we can obtain zero-order kinetics and, consequently, can express the activity in IUB enzyme units. It is possible that under the same conditions tyrosine release may be zero-order and could be measured kinetically, but the drawback is that tyrosine in the intact albumin substrate has similar physical and chemical properties to the free amino acid in the product matrix and it is necessary to separate, by physical means, the substrate and products. This is the procedure adopted in the classical methods for pepsin assay (5,6,10). The proposed kinetic method has the advantage of being carried out in homogeneous solution. Use of several systems of pepsin units precludes useful comparison of results from different centers; expressing pepsin activity in the same JUB enzyme units as are most other enzymes of clinical interest should facilitate comparisons of experimental and clinical findings. It is a widespread practice to use porcine pepsin as a reference standard in the absence of the availability of a pure human pepsin preparation, but kinetic methods of analysis do not require an external reference. Furthermore, we noted differences in behavior between porcine pepsin and human gastric juice pepsin, so that, in this system at least, porcine pepsin would not be a satisfactory standard. Clearly, human pepsin, when available, may be used to calibrate the present kinetic method. The converse is true also: this method may be used to compare the potency or degree of activity of any given pepsin preparation. Absence of interference from products derived from duodenal refiux or deliberately added substances such as phenol red is an important asset and, once the optimum is established, the analysis appears to proceed despite their presence. Standardization of pepsin assays in the manner we propose should allow large amounts of data to be collected and subjected to statistical analysis, which should illuminate this area of gastric pathophysiology. Pepsin dynamics are still poorly understood after decades of investigation (11) and this new method may help in their clarification. We acknowledge the help and provision of samples of human gastric juice from the Department of Experimental Gastroenterology, Royal Naval Hospital, Haslar, Gosport, U.K. Especial thanks are due to Professor R. H. Hunt, FRCP, Professor of Medicine at McMaster University Medical Center, Hamilton, Ontario, Canada, and Mr. T. Gledhill, FRCS, in the Division of Gastroenterology, McMaster, for valuable clinical discussions during the development of this paper. References 1. Samloff IM. Slow moving protease and the seven pepsinogens. 57, 659-669 (1969). 2. Taylor WH. Biochemistry of pepsin. In Handbook of Physiology, 5, Sect. 6, Alimentary Canal, CF Code, Ed., American Physiological Gastroenterology Soc., Washington, DC, 1968, pp 2567-2587. 3. Hunt JN. A new method for estimating peptic activity in gastric contents. Biochem J 42, 104-109(1948). 4. Wynn-Williams A. A simple method for the estimation of pepsin in gastric juice. J Clin Pat/wi 8,85 (1955). 5. Anson ML. The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin. J Gen Physiol 22, 79-89 (1938). 6. Burstad A. A modified hemoglobin substrate method for the estimation of pepsin in gastric juice. Scand J Gastroenterol 5,343348 (1970). 7. Cornish-Bowden A. Principles of Enzyme Kinetics, Butterworths, London, 1976, pp 25-27. 8. Ireland JT, Read RA. A thin layer chromatographic method for use in neonatal screening to detect excess amino acidaemia. Ann Clin Biochem 9, 129-132 (1972). 9. Colquhoun D. Lectures in Biostatistics, Clarendon Press, Oxford, London, 1971. 10. Klotz AP, Duvall MR. The laboratory determination of pepsin in gastric juice with radioactive-iodinated albumin. JLab Clin Med 50, 753-757 (1957). 11. Achord JL. Gastric pepsin and acid secretion in patients with acute and healed duodenal ulcer. Gastroenterology 81,15-18(1981). CLINICALCHEMISTRY, Vol. 29, No. 3, 1983 451