The Effect of Ultraviolet Light and Thiol Compounds on Guinea Pig Skin Histidase*

Vol. 46, No. 1

TRE JOORNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

Printed in U.S.A.

1966 by The Williams & Wilkins Co.

THE EFFECT OF ULTRAVIOLET LIGHT AND THIOL COMPOUNDS ON GUINEA PIG SKIN HISTIDASE* J. HILL ANGLIN, Ja., PID., DAVID H. JONES, M.S.,t ARLEY T. BEVER, PnD. AND MARK ALLEN EVERETT, M.D.

Total urocanic acid (tICA) content of skin Determination of Histidase Activity has been shown (1, 2) to be decreased in quanThe method used for the determination of histitity after irradiation with ultraviolet light UV dase was essentially that of La Du et at. (5), which in vivo. Trans UCA, the isomer normally pres- was adapted from the spectrophotometric proent in skin, is also partially converted to the cedure of Tabor and Mehier (6). This method is based on the fact that at 275 m the molar exisomeric cis form.

tinction coefficient of UCA is 18,000 between pH 7.2 and 11. Histidine has essentially no absorbance at this wavelength. Thus, as histidase changes histidine to [JCA the absorbancy at 275 m increases

Since UV alters UCA in skin, experiments were undertaken to determine if there was a UV effect on the enzyme forming UCA from L-histidine in the skin. This enzyme, designated histidase (EC 4.3.1.3) has been shown to deammoniate L-histidine 2-CTM producing UCA in guinea pig epidermal homogenates

due to the formation of UCA. Histidase activity was defined on the basis of an increase in optical densty (O.D.) per unit time.

(3). Histidase activity was shown to be present in epidermis but not dermis, while urocanase normally present in liver was absent in skin. Thus, in skin, the degradation of L-histidine is concluded after deanlmoniation and UCA ac-

The reaction mixture containing enzyme, substrate and GSH was buffered to pH 92, the pH optimum for histidase in vitro. The reactions were run at 25° C. Assay components were added in

the following order directly to the spectrophotom-

eter cuvettes: 0.1 ml of 2% skin homogenate or purified histidase fraction; 0.5 ml of 0.1 M pyrophosphate buffer (pH 92); 0.05 ml of 0.1 M GSH; 0.45 ml water; and 0.10 ml of 0.1 M L-histidine. The reaction was initiated by the addition of the

cumulates.

substrate, L-histidine, and the cuvette was immediately placed in the spectrophotometer.' The

EXPERIMENTAL PROCEDURES

change in absorbancy per minute was determined and this value was converted to jmoles UCA per

Preparation of 2% epidermal homogenate

in 0.1 M sodium prophosphate

minute per mg protein. Protein content was

determined by the method of Lowry et at. (7). buffer The specific unit of activity was defined as the Epidermal samples were obtained from white, mo1es of UCA formed per minute per mg of medium-sized guinea pigs (300—500 gm). A large protein. portion of the skin from the back was removed The blank consisted of the same mixture as the for collection of the epidermis by scraping (4). assay except no substrate was added. Then 100 mg portions of the epidermal scrapings were added to 5 ml of cold 0.1 M sodium pyroIrradiation in Vivo phosphate buffer, pH 9.2. The mixture was homog-

enized for approximately 15 seconds in all glass homogenizing apparatus' and centrifuged at 1500 X gm to remove the dense, insoluble material. The enzyme activity remained in the supernatant fraction. * From the Department of Dermatology and the Biochemistry Research Laboratory, University of Oklahoma Medical Center, Oklahoma City,

The animals were prepared for irradiation by clipping and shaving the backs with an electric razor. One side was shielded from irradiation for

National Institutes of Health, Bethesda, Mary-

Immediately after irradiation the animal was sacrificed and both the irradiated and control

the control.

The prepared areas were irradiated with a Burdick hot quartz mercury vapor lamp for 6

minutes at 20 inches from the source. The amount of UV light was determined to be approximately 6 X 1O ergs per cm'. This quantity of irradiation Oklahoma. t Present address: Department of Biochemistry, has been shown to be 3 times the amount needed to produce a barely perceptible redness (MED)' Cornell University, Ithaca, New York. § Present address: Division of Research Grants, after 20 hours. land.

This investigation was supported in part by

samples were prepared as homogenates and assayed research grants A-2344 and AM-05278 from the for histidase activity. In each animal tested the National Institutes of Health. Received for publication December 14, 1964. 'Cary Model 14 Spectrophotometer, Applied 'Duall Tissue Grinder, Kontes Glass Co., Vine- Physics Corporation. land, New Jersey.

5Minimal erythema dose.

34

35

GUINEA PIG SKIN HISTIDASE

TABLE I

control sample was assayed by a procedure identi-

cal to that used for the irradiated. Differences in activity found were attributed to the effect of UV

Purification procedure for skin histidase

on the skin.

Purification Procedure for Skin Hist'idase A study of the histidase reaction of a partially purified enzyme preparation was carried out. The first two steps of the purification procedure were adopted from the method of Yoskioka (8). A 25% homogenate was prepared in 0.1 M phosphate buffer, pH 8.0, from guinea pig epidermal scrapings and stirred for 1 hour at 0° C., pH 8.0, as an extraction procedure. 1) The mixture was then centrifuged at 1500 X gm. for 15 minutes and the supernatant retained. 2) To each ml of skin extract 0.025 g. of kaolin was added as an absorbing agent,

with 0.17 ml of 0.03 M thioglycerol employed to

preserve the —811 bonds. 3) The solution was heated at 65° C. for 8 minutes and centrifuged at

Purification step

Total Total Specific Activity fold YTld mg. units units increase protein tivity

1. Crude Ho- 488

2010

4.4

1170

8.26

100

mogenate

3. After Heating

4. After Acid

132

4.9

393

1.2

192 160

80.1

1.76 58.2 17.8

19.5

35.6

9.6

Prec. 5. (N114)2S04

Frac. 6. DEAE-Cellu-

025

36 1450

323

1.7

lose

Values = Specific units X 10' 20,000 X gm. for 20 minutes, and the supernatant min./mg protein X lo'

jsmoles UCA/

retained. 4) The solution thus obtained was

brought slowly to the isoelectric point of histi- tivities among the individual animals. For dase, pH 5.0, with 2.0 N acetic acid and centrifuged at 20,000 X gm. for 20 minutes with the precipitate

this reason, in subsequent irradiation experi-

being retained. The precipitated protein was re- ments, each animal was utilized as its own dissolved in 0.1 M phosphate buffer, pH 8.0. 5) control by comparison of the specific activity The material was retained which precipitated at found in bilaterally symmetrical areas of ir32—40% saturation of ammonium sulfate. This precipitate was redissolved in 0.1 M phosphate buffer, radiated and nonirradiated skin from the same pH 8.0. 6) The final step in the purification pro- animal.

cedure was the application of the enzyme solution in 5 ml volume to a 20 cm DEAE-cellulose colDelayed histidase activation with GSH umn. The protein solution was washed onto the Upon addition of GSH to skin homogenates column with 30 ml of 0.1 M tris-acetate buffer, pH 8.0. Elution of the protein was effected with a there was a delay of about 3 minutes before sodium chloride gradient starting at 0.2 M NaCI the reaction rate increased. The increased reacin 0.1 tris-acetate and going to 0.4 M NaC1. The tion rate remains linear for more than two samples were collected in 5 ml fractions. The histi-

dase activity was found in the fifth or sixth tube. The separation seemed to be sharp since the activity was eluted in 1 to 2 fractions. The partially purified histidase thus obtained was stored in the cold in the presence of thioglycerol as a protective agent. Calculations of increased specific activity and yield of initial enzyme activity are shown in Table I. RESULTS

Determination of normal histidase activity in guinea pig skin

The histidase activity was determined in the skins of 30 guinea pigs to establish an

hours under the conditions of the experiment. In the absence of exogenous GSH the rate is lower but linear (Fig. 1). This phenomenon was apparently unaffected by UV irradiation.

GSH effect on purified histidase

Partially purified histidase (1,600 specific units) was assayed in the manner previously described in an effort to gain more information

concerning the nature of the accelerated activity in presence of GSH found in crude enzyme reactions. The results obtained are shown in Figure 2. When the reaction was run in the absence of GSH, the rate was linear but slow. Upon addition of 4.2 tmoles GSH per ml the specific activity was increased about 7-fold after 3 min-

average histidase value. The mean value was 6.03 X 10 specific activity units with an average difference from the mean of 1.02 x utes. It may be seen in figures 1 and 2 that 10 specific units. These results indicated a the increase in activity following addition of wide range of variability of the histidase ac- GSH occurs after an interval of several mm-

36

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Effect of various thiol compounds on the histidase activity of partially purified histida.se

The effect of various thiol compounds on the activity of a sample of partially purified histidase was determined. The histidase employed was carried through the first 5 steps of the purification procedure omitting the Kaolin absorption of step 2. The enzyme preparation contained 26.6 units of specific activity when assayed in the absence of a thiol compound. The results are shown in Table II. L-cysteine is notable since it has an inhibitory effect in contrast to the expected activating effect of an -SH compound. Peterkofsky

5..

C,

z

0 0, 0,

and Mehler (9) have observed that cysteine serves as a very effective competitive inhibitor of the histidase from liver.

Effect of UV irradiation on histidase activity from skin 'I

5 6 1 8 9 0 II

2 18 I IS 6 Il IS

TIME (minutes)

After preliminary study of histidase activ-

ity in unirradiated skin preparations, the

Fia. 1. The effect of GSH on the delayed activation of histidase using epidermal homogenates:

1) Absence of exogenous GSH; 2) Same assay with 42 moles added GSH/ml.

utes. When the enzyme was preincubated with GSH before the addition of substrate, there was

no delay in the manifestation of accelerated activity (line 3 fig. 2).

GISH optimum of purified histidase

0,

5.. 5..

The GSH concentration which would pro- C,0, duce optimal activity in a purified histidase -50,0, preparation was determined. Increasing con- 00, centrations of GSH were added to aliquots of -5 the enzyme solution and each was assayed by the previously described method. Since this was a comparative study the results were tabulated as O.D. per 20 minutes instead of specific units. When these values were plotted

against tmoles GSH, a GSH optimum was found. The results are shown in Figure 3.

0123 56 75 9101112131'1151617 These data indicate that the optimum GSII TIME (minutes) concentration for the purified histidase is beFIG. 2. The effect of GSH on purified histidase tween 4 and 5 janoles per assay mixture (3.3— (1,830 units): 1) Assay run in the absence of 4.3 moles per nil). It can be seen from the GSH; 2) Assay run in the presence of GSH with graph that above this concentration a rapid de- no preincubation; 3) Assay run after 5 minute

crease in activity occurs.

preincubation with 5 ,amoles GSH. Substrate addition at zero time.

37

GUINEA PIG SKIN HISTIDASE

changes in activity of the enzyme were investigated following administration of UV in vivo.

The data from 10 such experiments are recorded in Table III. In every instance a higher specific activity of histidase was found in irradiated skin prepara-

TABLE II Effect of various thiol compounds on the histidase reaction Units

Compounds

tions when compared with nonirradiated skin Control from the same animal. The level of activity ex- Reduced GSH hibited a high degree of variability in the dif- Thioglycolic Acid

ferent animals. Analysis of the data by the Thioglycerol Wilcoxon Matched Pairs Signed Rank Test 2-thioethanol L-cysteirie

gave p < .01 uncertainty that the specific ac- Ethyl mercaptan tivity was greater in the irradiated skin (10). NailS UV irradiation effect on purified histidase in vitro

Values =

Specific

26.6 33.0 28.6 31.4 32.0

—

16.8

—37

32.2 38.0

21 42

24 8 19

20

units X 10

pmoles

UCA/min./mg protein X 10'.

Control, no added thiol, S ,zmoles of each comIn a series of irradiation experiments, 1 ml pound added to assay mixture. solutions of purified histidase (1,600 specific units) were subjected to a monochromatic TABLE III beam of 285 me light with an intensity of 5.6 t Effect of UV irradiation in vivo on

watts per cm2 per second for varying time

intervals. These experiments were performed to investigate the possibility that the activation

observed in vivo was produced by a direct

Control

Irradiated

1

6.1

6.3

.200 .180.

10

3.1 5.3 4.4 5.8 8.2 5.9 3.2 5.4 8.4

4.3 8.5 4.8 6.3 9.9 8.6 3.8 5.7 8.7

.260

.2l0. .220.

z

Histidase units Ssmple

2 3 4 5 6 7 8 9

.280

.e

skin histidase activity Increase of irradiated over control

.2 1.2 3.2 .4 .5 1.7 2.7 .6 .3

.3

Values = Specific units X 108 = zmoles UCA/ min./mg protein X 10'.

.160.

.I'lO. .120.

effect of UV on the enzyme. The enzyme

.100

preparations suspended in 0.1 M phosphate

.080

buffer, pH 8.0, with no added thiol compound,

were placed under the UV source. Aliquots

.060

.020 .050

5

0

5

20

I

I

25

80

I

85

I

80 q 50 55 80

sItOles OSII

Fie. 3. Effect of GSH on partially purified histi-

dase (2,180 units) showing change in enzyme activity with different concentrations of GSH. The ordinate represents the enzyme activity measured as change in optical density/20 mm. at 275 mo.

were removed at intervals and assayed by the above procedure. Inactivation of the enzyme was observed. The experiment was repeated several times, and data from a typical run is shown in Figure 4 where z O.D./20 min. has been plotted against time. DISCUSSION

Urocanic acid constitutes about 80% of the UV absorbancy of dilute acid epidermal cx-

38

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

It has been shown (6) with enzyme isolated from liver, that reduced sulfide groups (—SH) are required for full histidase activity. In addi-

Ltion Ichihara (12), observed that sulfhydryl enzyme inhibitors will inhibit the histidase reaction, thus indicating that at least part of the sulfide groups must be in the —511 state for full

activity. A number of thiol compounds were employed in this study and all except cysteine caused activation. L-cysteine apparently functions in some role other than that of a

f. 0,

0,

donor. In fresh homogenates the enzyme exhibits a

fair amount of activity even in the absence of added GSH. However, with purified enzyme preparations, very little activity was observed in the absence of exogenous GSH. The results

0 •1

obtained in this study appear to agree with the previous observation that some of the 5

0

IS

20

5

.00

TIME OF EXPOSURE TO U. (minutes)

Fia. 4. Effect of increasing UV irradiation on aliquots of a partially purified histidase solution. The ordinate represents the enzyme activity measured as change in optical density/20 mm. at 275

mgi.

tracts (11). This suggests that UCA serves to protect the deeper layers of the skin from the harmful effects of UV rays striking the epidermis. The function of histidase in skin may be to maintain a fixed level of UCA,

since it appears that the UCA present is

sulfide bonds functioning in the active sites must be in the —SH form. The immediate increased activity observed in purified enzyme (Figure 2 line 3) implies that the function of GSH is to react with the enzyme and not substrate, possibly modifying the necessary groups on the enzyme so that maximal activity is observed immediately upon contact with substrate. A rather wide variability of histidase values was found when the specific activity level was determined in skin from 30 individual animals. However, under identical conditions of assay an increase in specific histidase activity was found in the skin of each animal tested when assayed

formed entirely by the enzymatic deammonia- immediately after irradiation and compared tion of histidine. The initial increased histidase with the activity found in unirradiated biactivity following UV exposure tends to mini- laterally symmetrical skin. mize the decrease in UCA observed (2). The SUMMARY primary photochemical events leading to inTTV irradiation of guinea pig skin in vivo creased histidase activity have not been deter-

mined by these experiments, although it has produces an increase in epidermal histidase been demonstrated that an increase in activity activity. Irradiation in vitro does not. Thdeoccurs only in vivo and not in crude homoge- pendent of UV irradiation effect, GSII and nates or purified enzyme solutions.

several other thiols increased the rate of enzyme

Possibly some of the enzyme exists in skin activity. L-cysteine decreases the activity. An as a "pro-enzyme" which has the active site optimum GSH concentration is shown above

unmasked by a photochemical effect. This which the activity is decreased. would account for the failure of the purified REFERENCES preparation to exhibit an increase in activity upon exposure to UV. Conditions existing in a homogenate may be sufficiently different from those in situ to prevent release of appreciable amounts of the active form of the enzyme.

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GUINEA PIG SKIN HISTIDASE

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