Direct spectrophotometric assay for reactions producing small pH changes

ANALYTICAL

38, 21&223

BIOCHEMISTRY

Direct

(1970)

Spectrophotometric Producing

Assay Small

CHRISTIAN Harvard

School

of Dental Boston,

pH

for

Reactions

Changes

SCHWABE

Medicine Massachusetts

Received April

and

Harvard 02116

Medical

School,

14, 1970

Many reactions, enzymic or nonenzymic, may be quantified by measuring the pH change occurring during the conversion of reactants to products. Although in a vast majority of cases glass electrodes are used for this purpose, several shortcomings make them unsuitable for specific applications. They are slow to respond, are slow ,to equilibrate if rapid temperature changes are desired, and are subject to large changes in junction potential when proteins are adsorbed on the glass membrane. Indicators which change their color as a function of hydrogen ion concentration react instantaneously and have therefore found applications in stopped-flow techniques in conjunction with calorimeters (1). Many of these visible indicators are in fact protein dyes. In this paper we are reporting the use of sodium diethylbarbiturate (sodium barbital) for the spectrophotometric detection of minute pH changes in solutions. The molecule does not appear to absorb to any extent on the proteins tested and the changes of its pK value as a function of ionic strength and temperature are small and predictable. The ionized form of sodium barbital has a reasonably high molar extinction value at a wavelength between the peptide bond adsorption and the nucleic acid peak (2400 ,to 2500 A). The change in optical density (AOD) per pH unit at 2400 to 2500 A is very large and linearly dependent on the indicator concentration in the useful range (1F to 1O-32M) in a 10 mm light path. The useful pH range for sodium barbital extents from 7.5 to 8.5. Barbituric acid serves similarly at acid pH values but possesses a higher molar extinction at 2570 A. Application of this indicator as a means ‘to detect and to quantify peptide hydrolase activity and protease activity are demonstrated in this paper. Other applications are discussed. The optical pH measurements of solutions of small molecules may ’ NIH

Career Development

Awardee

No. K3-DE-11,128. 210

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be extended to pH 11.0 by the addition of 1O-4 M phenol or RP4 M tyrosine to an equimolar solution of sodium barbital. Some useful properties of this combined indicator solution are also presented in this paper. MATERIALS

N-Ethylbarbiturate, barbituric acid, and phenol crystals (AR) were purchased from Fisher Scientific Co. and used without pretreatment. Leucylleucine (chromatographically pure), oxidized RNase A, and ultra pure urea were obtained from Mann Research Laboratories and tyrosine (chromatographically pure) from Schwarz BioResearch. All spectrophotometric work was performed with a Gary model 15 outfitted with an automatic sample changer. STANDARDS,

PRINCIPLES,

AND

LIMITATIONS

In this section the most important characteristics of the ultraviolet pH indicator concerning the principle of their application to optical pH measurements are reported. Absorption

Spectra

and Molar

Extinction

Upon loss of a proton, sodium barbital acquires a strong absorption band at 24008. Tyrosine and phenol similarly give rise to a strong #absorption band near 24OOA upon ionization of the hydroxyl group. The apparent pK’s of these compounds are *at pH 8.1, 9.8, and 10.0, respectively, for sodium barbital, tyrosine, and phenol. Barbituric acid hlas a pK at pH 4.0 and gives rise to a similar spectrum at acid pH values (peak at 2570A). The first three compounds may be used singly or together depending upon the extent of the pH change anticipated for a certain reaction. The relatively low pK value of barbital makes it more suitable for the measurement of enzymic activity. The molar absorption value for sodium barbital in its basic form is 9.7 X lo3 (20”, 10 mm light path). Beer’s law is valid over all useful concentrations of the indicator (10-5 to 1e3 M). The ,optical density change corresponding to a given pH change may therefore be varied as desired by a change in the indicator concentration. With the laid of Fig. 1 the change in OD per change in pH that is most desirable for a given experimental condition may be predetermined. Obviously a compromise must be reached between the sensitivity attainable and the background absorption. Standard

Curves

While under closely controlled conditions absolute pH measurements are possible with this system, its greatest utility lies probably with the

212

CHFtISTIAN

SCHWABE

0.8 0.7 1 2

0.6

s

0.5 z 0.4

8 Q

0.3

2

4

6

VERONAL

8

IO

CM) x IO4

FIG. 1. Increase in optical density (AOD) at 24508 for each 0.1 pH unit depicted as function of sodium barbital concentration. The shaded area indicates increased sensitivity to pH changes with increasing indicator concentration. The lower line represents background absorption. If, for example, an experimental condition requires a starting pH of 7.3 and should be observed up to pH 7.4, a 6 X 16* M Verona1 solution (OL = 1.32) could be used. The AOD for 0.1 pH unit would be 0264, leading to a final OD of l&34. The same reaction at an initial pH of 8.1 would require a lower Veronal concentration (a 6 X lO-'M Veronal solution at pH 8.1 absorbs 3.0 OD units).

measurement of pH changes in a solution. The sensitivity of this measurement depends upon the slope of the linear portion of a plot of optical density versus pH, which in turn depends upon concentration of the indicator. Since neither primary standard sodium barbital, phenol, nor tyrosine is commercially available, the indicator solution must be standardized by each investigator to determine the slope of the pH change versus the change in optical density. This procedure is similar to the standardization of electrodes with a standard buffer. In order to determine the slope and the linearity of the optical density change as a function of pH for the mixed indicator as well ‘as sodium barbital alone, an experimental setup was used which combined the Radiometer pH-stat titration assembly and pH meter 26 with a Cary 15 spectrophotometer. Utilizing the data ,depicted in Fig. 2, the relationship (between optical density and pH of the mixed indioator
+ 6.8 (245Ori)

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I

1.8 _ 1.716. 1.5. 1.4 13_ ? E T N ,” 2 m E =: 2

1.2 _ I.1 _ 1.0 0.9oa07_ 0.6

_

0.50.4

_

03_ 0.2

_

0.1

-

, 5

6

7

8

I 9

IO

II

12

PH

FIQ. 2. Titration of sodium barbital (Veronal)-tyrosine-phenol mixture (lOA M with respect to each compound). Strong base (0.1 N NaOH) was added in fractions of a microliter through manual operation of the micrometer screw of the Radiometer tit&ion assembly. The points were obtained from experiments performed several days apart.

from which follows that:

for the linear position of the curve, where c is the molar concentration of each of the indicators present (i.e., if the solution is 10-*&f in each (sodium barbital, phenol, and tyrosine), then c = lOA). Since the optical density of the indicator solution is linearly dependent upon indicator concentration, titration curves obtained at various indicator concentrations should all extrapolate back to pH 6.8 (equation 1). In order to standardize the indicator solution it is therefore sufficient to measure the optical density of such solution at a pH which falls into the linear range and to draw a straight line from pH 6.8 through this experimental point.

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CHRISTIAN

SCHWABE

The slopes thus obtained will represent the relationship between optical density and pH as long as measurements are carried out in the range in which equation 1 is valid. Similarly, if measurements were made at a wavelength other than 2400 or 2450A, the linear portions of the standard curves obtained should still extrapolate back to pH 6.8 (see Fig. 3). These properties of the indicator afford a very expedient way for the standardization, sufficiently precise for most applications.2

09Of30.7 _ 0.6 0.5 0.4 0.30.2

t

01; 1

60

I.1 C’

65

70

!

I

7.5

80

11

85

90

PH FIG. 3. Titration of a lOA M sodium barbital solution at the wavelength indicated in the figure. The linear portions of the titration curves converge at pH 6.3. Beer’s law is valid for measurable concentrations at every wavelength within the adsorption band (-230-260 ma).

Sensitivity

In order to consider a method for certain applications it is imperative to know its limitations. The sensitivty of this method clearly depends upon the quality of the spectrophotometer ‘available. Many instruments marketed today permit measurements up to three OD units. This is very important since the sensitivity of this method increases with the indicator concentration, i.e., with the increased optical density of the indicator solution. A 3 X 1O-4 M sodium barbital solution (OD 1.5) ‘It is understood that the relation of OD to pH is not exactly linear since the optical titration curves can be described by a Henderson-Hasselbalch equation. The absence of a cooperative effect (which produces the sharp end-point with phenolphthslein, for example) makes the straight-line approximation for a finite pH range useful in the case of sodium barbital. For most exacting work a narrower pH range should be used.

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would indicate a pH change of 0.1 unit as Ian OD change of 0.122. Since 0.01 OD unit represents a 10% scale deflection of the Cary 15 (using an expanded scale), a pH change of 0.01 is easily measured. The sensitivity and stability
Ionic strength changes up to 0.1 have a negligible influence on the ionizati,on of sodium barbital. Thereafter the change in optical density due to neutral salts becomes a signifioant factor if absolute pH measurements are desired. The influence on aOD values remains very small. The effect of salt on any other buffers present in the solution will indirectly affect the indic,ator and must be taken into consideration. In a typical experiment the influence of KC1 on the ionieation of barbital at pH 8.2 was measured in the presence of Tris and changes of pH were compared with OD values. The relsation was linear up to 0.2 ionic strength, after which the direct effect of the salt on the pK of sodium barbital beoame apparent. Interaction

with Proteins

The adsorption of the sodium barbital indicator on protein molecules could conceivably lead to a change in its pK value and alter its specific absorption without an actual change in hydrogen ion concentr,ation. Since all proteins have different properties, no general statement can be made about this aspect of the procedure. The following experiment was done with crystalline bovine serum albumin and 1O-4 M sodium b,arbital in the presence ,of 0.01 M KCl. Bovine serum albumin in concentrations of 0.1, 0.5, 1.0, and 2.0 mg per ml were mixed with an equal volume of 2 X 10e4 M sodium barbital indicator (0.02 M in KCI) . The optical density reading at 245OA for all these solutions indicated pH values identical to those obtained ‘by electrodes when compared with the standard pH curve for lo-*M barbital. Any interaction of sodium barbital with bovine serum salbumin either is negligible or does not give rise to any erroneous pH readings in the spectrophotometer. If the barbital. system is used with unknown protein solutions, an occasional reference check with a good pH meter is necessary to assure that the protein present in the solution did not alter the optical properties of the indicator. These checks ‘are particularly important if a crude tissue extract is assayed, since such preparations usually contain nucleic acids or other cellular elements which might interact strongly with sodium barbital.

216

CHRISTIAN

Reactions

Producing

SCHWABE

Buffering

Species

With the {barbital method, the measurement of all reactions leading to an increase in net charge (ester hydrolysis or COz hydration, for example) are easy to interpret; but to quantify a reaction in which buffering species
SECTION

AND RESULTS

A few selected experiments are reported in order to illustrate practical applications and to give guidelines for adaptation of the method to other assay systems. Hydrolysis

of Dipeptides

by Leucine

Aminopeptidase

A 5 mM solution of Leu-Leu was hydrolyzed at 40’ by varying amounts of a purified leucine aminopeptidase from fibroblasts (2) on a Car-y 15 spectrophotometer (0 to 1 .O OD scale, 2450 A), utilizing #a Cary sample changer (Fig. 5). The lag phase is particularly apparent at low enzyme concentrations. Since the indicator used was lOA M, the total OD change (0.56 unit) reflects an increase of the starting pH (7.8) to pH 9.1. Because it is impractical and undesirable to observe reactions to their

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88 8.7 8.6 85 84 PH

83 82 8 I 80 79 78 MOLES

OF SUBSTRATES (x 1041

HYDROLYZED

FIG. 4. Hydrolysis of leucylleucine of the following millimolar concentrations: 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0 for line a, b, c, d, e, and f, respectively. A horizontal line through the graph at any value would cut the curves at points where equal per cent hydrolysis has occurred for all concentrations of substrate. The lines were calculated based on a pK, of Leu-Leu corresponding to pH 7.5 and an original pH of 7.8 for the reaction mixture. The points were averaged from at least three experiments.

completion, a fast and reliable method for routine assays is required. The “pH jump” method can be employed without complication by buffering species present. A known equivalent of acid is added to the reaction mixture directly into the cell and stirred rapidly with a plastic plunger. Since sodium barbital absorbs less at Ia lower pH, the OD reading will drop as a consequence of the acid addition. The time required to reach the original density is measured as distance on the synchronized motor-driven recorder chart. If more buffering capacity mnas present in sample A compared to sample B, A will show a less pronounced drop in OD with the addition of acid, hence less optical density has to be recovered. All factors otherwise affecting the pH (OD) change in the reaction mixture are contained in one constant (i.e., the amount of acid added) and the recovery to the original OD is solely a function of activity and time. If one proton is consumed per bond hydrolyzed it is most convenient to add acid corresponding to a known percentage of substrate equivalents present. One might thus predetermine the pH step produced to correspond to 10% hydrolysis of the substrate (Fig. 6). The total measured pH ch,ange in this experiment varied from 0.1 to 0.2 pH unit. Small amounts of relatively strong titrant should be used to avoid

218

CHRISTIAN

SCHWABE

TIME Fm. 5. Leucylleucine solution (5 mM) hydrolysis with the following amounts of leucine aminopeptidase: (1) 6 pg, (2) 12 gg, (3) 24 pg, (4) 48 pg. C = control (without enzyme). In this case rates were directly comparable because the substrate concentration (buffering species) was the same in every cuvet. Every solution was measured every 66 seconds using an automatic sample changer in Gary 15 spectrophotometer.

dilution of the indicator. Alternately the acid or base can be prepared in sodium barbital. A 250 ,yl gastight Hamilton syringe with a dispensing head delivers 5 ~1 & 1% error. The titrant is quickly mixed in the cell by a plastic plunger. Activity

of Leucine

Aminopeptidase

in Urea Solutions

The utility of the barbital indicator assay method under ‘adverse conditions is demonstrated #by the following experiments. The purified connective tissue LAP ,appears to be stable in 8M and 10 M urea at 40” for 24 hours (3). To test whether the enzyme retained ,activity in urea, solutions were made (2, 4, 6, 8, 10 M urea) containing lo-’ sodium N-ethylbarbiturate (pH 7.8) and 2.5 X 1O-3M Leu-Leu substrate. The solutions were pipetted into the UV cuvets of the Car-y 15 ,automatic sample changer and equilibrated to 40”. A slight positive

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TIME FIQ. 6. (a) Original recording of hydrolysis of a 2.5 m&f solution of leucylleucine by four different amounts of connective tissue leucine aminopeptidase. At time A each cuvet contained indicator plus substrate. At time B the proper amount of enzyme was added to each cuvet and at time C, 5 ~1 of 0.05 N HCl. The dotted lines show the pH jump for every cell and the solid lines indicate the point at which the original pH (OD) was again achieved. A short section of the slope has been marked for clarity. The cuvets contain 1.0, 2.20, 3.30, 4.40, and 5.60 pg of enzyme. Since the enzyme is stored in Tris-HCI, an increasing amount of buffer was added with increasing amounts of enzyme, and the pH jump produced was progressively less. At the two highest concentrations the substrate begins to limit the reaction velocity. In (b), the same amount of enzyme was added to differing amounts of leucylleucine. The cells containing the total system are designated B and the same cells after the addition of acid as A. The pH jump produced by 5 gl of 0.05 N HCI is of different magnitude in the various cells according to the substrate concentration. In samples 3, 4, and 5, the original pH is recovered at the same time as expected from theoretical considerations (see “Standards, Principles, and Limitations”). In cells 1 and 2 the pH jump produced was too extensive. It exceeded the amount of buffering species (substrate) present, emphasizing that equivalents of acid added must be a small fraction (-10%) of the total number of equivalents of base released during the reaction, This experiment illustrates the fact that in this system (within limits) the buffer concentrations may be ignored if the reaction is permitted to recover exactly the original OD.

220

CHRISTIAN

SCHWABE

pressure of Nz was maintained in the sample chamber to avoid COZ interference. The reference cuvet contained sodium barbital in Tris (pH 7.8) and substrate (2.5 x Ws M Leu-Leu). The reaction was started by the addition of 10 ~1 of leucine amin,opeptidase (2 mg/ml) to the total of 1 ml reaction mixture. After one OD reading (220 see) a pH jump (0.15 unit) was effected by addition of 5 ~1 of 0.05 M HCl. The result depicted in Fig. 7 shows that the enzyme is active even in 10 M urea. The slowdown of the reaction may be due to the fact that water is no longer the only species in the vicinity of the active site but that one out of three encounters must bring an urea molecule instead near the enzymesubstrate complex. The retention of activity under the above conditions could qualitatively be demonstrated by thin-layer chromatography.

100

1

I

95

90 mole

I

85 fraction

80

75

70

of H,O

Fro. 7. Activity of leucine aminopeptidsse in urea solutions. A 2.5 mM solution of leucylleucine was hydrolyzed at 40” in the presence of 10”M sodium barbital. The time required to recover 0.25 heq (10% of the substrate) was measured and converted and the rate expressed as pmoles leucylleucine hydrolyzed per minute per milligram, assuming 1 proton to be consumed per bond. The rate of the reaction was plotted against the approximate mole per cent of water in the urea solutions. The lack of discontinuity in this curve suggests that the enzyme is essentially untiected by the urea and that the reduced activity is probably due to the decreased water concentration.

H~C~TO.!YS~Sof Oxidized Ribonuclease by Trypsin ,Oxidized ribonuclease (2 mg/ml) was dissolved in *a mixed indicator solution (UY4 M each sodium barbital, phenol, and tyrosine), 0.2 M in KCl. The pH was adjusted to 8.5. The reference cuvet received water,; a control cuvet received indicator and substrate, but no enzyme. The automatic sample changer was adjusted to read two of the five cells in the turret every 2.5 minutes. Figure 8 shows a plot of the hydrolysis of RNase by 10 pg of crystallized tryspin. In constrast to the peptide

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10 15 20 25 30 35 40 TIME (MINI

8. Hydrolysis of 2 mg ribonuclease by trypsin method. No buffer was added to the system.

monitored

by the sodium

hydrolysis in this system, a drop of the pH toward the isoionic point of the newly produced peptides occurs. If desirable, the pH change could be limited to less than one unit by any buffer system in the reaction mixture without a significant sacrifice of sensitivity. While it is difficult (with any method) to quantify proteolysis meaningfully, this method can conveniently be used to determine the extent to which a protein has ‘been digested. DISCUSSION

The chemical properties of a dipeptide and its hydrolysis products are not very ,different. A twofold increase in free carboxyl groups or in primary amine groups can maximally be attained if the substrate is completely hydrolyzed. This is a severe handicap for most methods except for a lately developed ninhydrin procedure (3). All methods involve more manipulation than the one described here. Immediate and continuous readings are an additional advantage of the direct spectrophotometric method. Artifacts have not been observed during our studies. In contrast, initial attempts to measure this reaction on a pH-stat failed because subtle differences were not detected and titration curves were ocoasionally obtained which were unrelated to the reaction. Particularly the peptide bond hydrolysis in urea could not be performed with electrodes. A potential source for artifacts lies in the specific interaction of the indicator with other solute molecules which could lead to a change in the pK value of the l’atter, i.e., simulate a change in hydrogen ion activity. While sodium barbital and barbituric acid appear to have very little affinity for pure proteins, phenol and tyrosine (through their hydrophobic centers) are more likely to interact. This aspect of the problem has severely limited attempts to optically measure pH with indicators in the visible range. A thorough investigation of the ultraviolet

222

CHRISTIAN

SCHWABE

indicators and an intense search for other molecules to better serve this purpose are indicated. It should be emphasized that changes in pH (APH) are measured in spite of possible #adsorpti,on phenomena. The activity of leucine aminopeptidase measured by this procedure showed results identical to those obtained by the most careful ninhydrin assay. It is significant that in this system (as in the BSA experiment) the absolute pH values detected optically at the beginning and the end of the assay corresponded to the electrodically measured values. Any reaction system exhibiting a pH change but not involving compounds with strong absorption bands between 2400 and 2600A can Ibe readily measured by this method.3 Several assays, not reported here, have been successfully performed, such as CO, hydration and the peptide and ester thydrolysis at extremely low substrate or enzyme concentrations. The UV indicator method in conjunction with the “pH jump” procedure requires a finite pH chlange to occur. While this is of no serious consequence for even very accurate work (pH optimum of enzymes are usually broader than the 0.1 pH jump), it would seem desirable for some reactions to be observed optically cat constant pH. For such purposes the method can be the basis for an optical pH-stat. Preliminary work in this direction has been successful. The method thus differs in principle and applicability from a direct spectrophotometric method (4) for the detection of esterase activity. SUMMARY

The hydrolysis of a dipeptide at pH 8.0 causes a shift in hydrogen ion concentration due to the fact that the a-amino groups of most amino acids are stronger bases than the N-terminal amino groups of peptides. The increase in pH, caused ‘by the hydrolysis of a dipeptide, is small even in an unbuffered solution, but the method here described is sufficiently sensitive to detect changes of 0.01 pH unit with a good spectrophotometer. The method is fast and #accurate once standard conditions are established. Very small amounts of enzyme are required at substrate concentrations much lower than ordinarily used. Since no electrodes are involved, neither changes in the junction potenti,als due to protein absorption nor temperature coefficients need to be considered. This method was used to measure the activity of connective tissue leucine aminopeptidase in 8 and 10 M urea. *For the pH range 3.5 to 4.5 barbituric acid must be used. Sodium barbiturate, phenol, and tyrosine are applicable for the pH range from 7.5 to 10.5. With proper precautions, the nonlinear region can be used to extend the range of this method.

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ACKNOWLEDGMENT This work was supported in part by a grant from the Life Insurance Research Fund and NIH Grant 3ROl DEO2453-0251.

1. 2. 3. 4.

FINLAYSON, SCHWABE, MATHESON, HUMMEL,

REFERENCES B., AND TAYLER, E. W., Biochemistry 8, 802 (1969). C., Biochemistry 8, 783 (1969). A. T., AND TATTRI, B. L., Can. J. Biochem. 42, 95 (1964). B. C. W., Can. J. B&hem. 37, 1393 (1959).

Medical