PURIFICATION and CHARACTERIZATION of RIBONUCLEASE from PSORIATIC SCALES* MAURICE LISS, Pn.D

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PURIFICATION AND CHARACTERIZATION OF RIBONUCLEASE FROM PSORIATIC SCALES* MAURICE LISS, Pn.D. AND WALTER F. LEVER, M.D. Psoriatic scales and psoriatic epidermis, in30 minutes and then centrifuged. The optical density of five-fold aqueous dilutions of the comparison with callus and normal epidermis,supernatant solution was determined in a contain relatively large amounts of both RNABeckman DU spectrophotometer at 260 mp and and DNA (1—3). In addition, various hydrolytic280 mp using a ten-fold aqueous dilution of Mac- products of RNA have been detected in psoriaticFadyen's regent as the blank solution. When the scales (2, 4). Santoianni and Rothman haveextent of hydrolysis of RNA was determined also by phosphorus analysis, 2 ml of the supernatant shown in biochemical studies that human epi-solution were used. Enzyme, substrate and zero dermis possesses RNase activity (5). Steigledertime controls were done in the same way. and Raab, in histoehemical studies, have re- Protein Determination. Protein was determined cently reported that the RNase activity in theby the method of Lowry et. at (11) or of Warburg horny layer of psoriatie lesions was stronger thanand Christian (12). Determination of the Nucteotide Composition of in the normal horny layer, and was present inRNA Before and After Prolonged Digestion with the entire stratum corneum (6). Psoriatic RNase. Ten mg of purified yeast RNA In this paper, we wish to report on the isola-were incubated with 0.5 mg of purified psoriatic tion and characterization of RNase from psori-RNase for 3 hours according to the standard pro- atic scales. The enzyme was found to be presentcedure. The incubation mixture was then dialyzed overnight at 4° against frequent changes of dis- in high concentration and to have a very lowtilled water. The dialyzed material ("core" RNA) molecular weight. was hydrolyzed with 0.15 ml of 70% HC1O4 for one hour at 100°. The acid hydrolysate was diluted MATERIALS AND METHODS with water and centrifuged. Aliquots were then spotted on Whatman No. 1 filter paper (13). The Preparation of ENA. Purified RNA was pre-chromatogram was run for 36 hours using the iso- pared from bakers' yeast according to the pro-propanol-HC1-water solvent system described by cedure of Crestfield, Smith and Allen (7). Wyatt (14). The ultraviolet light absorbing purine Phosphorus Analysis. Phosphorus was analyzedand pyrimidine compounds were eluted with 0.1N according to a modification of the procedure ofHC1 for 2 hours at room temperature and examined Fiske and SubbaRow (8) in which the reducingin a Beckman DU speetrophotometer. Adjacent agent was a 1% solutiun of mono-methyl-p-amino- areas of equal size were treated in the same way phenol sulfate (Elon, Eastman Kodak Company)and served as blanks. The compounds were identi- in 3% NaHSO3. fied by their relative rates of chromatographic Standard ENase Assay. RNase was assayed ac-migration and by spectral analysis. The com- cording to the procedure of McDonald (9). One ml pounds were quantitated using the following of substrate solution containing 10 mg of RNA inmolecular extinction coefficients: adenine, 12.6 X 0.1 M sodium acetate buffer of pH 5.0 was incu-10 at 262.5 met; guanine, 11.1 X 10' at 249 mp; bated for 15 minutes at 37° with a suitable aliquotcytosine, 10.0 X 10' at 276 met; and uracil, 8.15 X of enzyme solution. The final volume was adjusted 10 at 260 mji (13). The base composition of puri- to 2 ml with 0.1 M sodium acetate buffer. The re-fied yeast RNA before treatment with psoriatic action was stopped by the addition of 2 ml ofRNase was also determined by the HCI 04 hydroly- MacFadyen's reagent, consisting of 0.25%sis procedure. uranium acetate in 2.5% trichloroacetic acid (10). Starch Gel Elect rophoresis. For starch gel eleetro- The suspension was left at room temperature forphoresis the discontinuous borate buffer system described by Barret, Friesen and Astwood (15) *Fromthe Dermatology Research Laboratorieswas used. Starch gel was prepared using hy- of the Boston Dispensary and the Department ofdrolyzed starch (Connaught Medical Research Dermatology, Tufts University School of Medi-Laboratories). The gel buffer contained 90 volumes cine, Boston, Massachusetts. This investigation was supported by Researchof 0.016 M Tris-0.0033 M citric acid and 10 Grants No. A-5931, A-4486 and Graduate Training volumes of 0.02 M lithium hydroxide—0.076 M Grant No. 2A-5220 from the National Instituteboric acid. The final pH was 8.0. The electrode of Arthritis and Metabolic Diseases, United Statesvessels contained a lithium borate buffer (0.1M Public Health Service. Presented at the Twenty-third Annual Meetinglithium hydroxideM.38 M boric acid) of pH 8.0. of The Society for Investigative Dermatology,Filter paper strips (either S & S No. 470 or What- Inc., Chicago, Ill., June 28, 1962. man No. 3), 6 x 10 mm, were moistened with the 529 530 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY

RNase solutions being studied and inserted intowas liberated. The ribonuclease activity in the starch gel 22 cm from the cathode. The gelpsoriatic scales was at least 3 times greater than was cooled to —5° during the electrophoresis and a constant amperage of 60 mamp. was maintained. that reported for normal human epidermis by The voltage was increased intermittently duringSantoianni and Rothman (5). electrophoresis in order to maintain a 60 mamp. Purification of Psoriatic fiNase. Pooled samples current since resistance increased with the ad-of psoriatic scales were washed with distilled vance of borate ions. The electrophoresis was run for approximately 3 hours, at which time thewater at room temperature and acetone powders borate "front" had advanced 24 cm from thewere prepared at 4°. Twenty seven grams of the cathode. The protein bands resolved by electro-acetone powder preparations were extracted with phoresis were visualized by staining with water-500 ml of 0.25 N 112S04 for 2 hours at 4° with soluble nigrosin (15). shaking. The acid mixture was allowed to stand Preparation of DEAE-celtulose. DEAE-cellu-in the cold overnight and was then reshaken for lose (Brown Company), type 40, analytical grade, was prepared in a batch process by treatment withan additional hour. The material was centrifuged a 0.2 M NaOH solution containing 0.5 M NaC1at 4,000 r.p.m. for 30 minutes at 4°. The sediment and followed by removal of excess alkali with dis-was washed with 150 ml of cold 0.25N H2S04. tilled water. The ion exchange cellulose was then stirred with a 1.5 M NaHCO3 solution, filtered, andrecentrifuged, and the supernatant solutions finally equilibrated by successive treatmentscombined (Stage 1). The combined supernatant with a 0.05 M NaHCO3 solution. solutions were brought to 0.65 ammonium sulfate saturation (430 g of (NH4)2 504/liter) and the EESULTS material stirred in the cold for one hour and then RNase Activity in Psoriatic Scales. Acetonecentrifuged at 4,000 r.p.m. for 30 minutes at 4°. powders of psoriatic scales were extracted withThe supernatant solution was brought to 0.8 0.25 N sulfuric acid according to the procedureammonium sulfate saturation (105 g of (NH4)2 described under "Purification of PsoriaticS04/liter). The turbid solution was stirred at 4° RNase". The acid extracts were neutralized withfor one hour and then allowed to stand in the a dilute NaOH solution and the sediment re-cold overnight without stirring. The fiocculent moved by centrifugation. Determination ofprecipitate was recovered by centrifugation at ribonuclease activity in the neutralized acid4,000 r.p.m. for 30 minutes at 4° and the super- extracts revealed a release of acid uranyl acetate-natant solution discarded. The precipitate was soluble nucleotides with a concomitant release oftaken up in 10 ml of distilled water and turbidity almost equimolar amounts of organically boundwas removed by centrifugation at 18,000 r.p.m. phosphorus (Fig. 1). No inorganic phosphorusfor 30 mm. at 4° (Stage 2). The enzyme solution was heated at 90° for 3 mm. in a water bath and IA TABLE I 1.2 Purification of Psoriatic RNase CL0 The assays were carried out as described in the 'ii text. 0 -J Treatment Enzyme Tjnits° Specific 0.6 Activityt C-J - 0.4 Stage 1 (112504) 5,070 7 Stage 2 (0.65—0.8 AS.) 896 59 0.2 Stage 3 (90°) 603 71 Stage 4 (0.8 AS.) 386 81 Stage 5 (DEAE) 23 177 10 20 30 40 50 60 70 80 90 MIN. * Oneenzyme unit is that amount which will Fra. 1. Ribonuclease activity of neutralized 0.25 Nliberate 0.1 ,zmoles of nucleotide per incubation sulfuric acid extracts of psoriatic scales. Assaysmixture. An optical density of 10 at 260 mp was were performed according to the standard procedure. Solid circles represent organically boundassumed to be equivalent to 1 mM uranyl acetate- phosphorus. No inorganic phosphorus was re-soluble nucleotide (16). leased. tUnitsper mg of protein. PSORIATIC RNASE 531

4 = 1.6X 10—' FIG. 2. Sedimentation characteristics of psoriatic RNase at Stage 4 of purification (cf. Table I) in 0.02M Tris-HC1 buffer of pH 7.2 containing 0.1M NaC1. The protein concentration was 7 mg per ml; temperature, 4°; speed, 59,780 r.p.m.; bar angle, 60° for pictures (1) and (2) and 50° for pictures (3)— (10). Photographs were taken at 16-minute intervals after reaching maximal speed.

immediately cooled by immersion in an icein a Spinco Model E analytical ultracentrifuge. bath. The flocculent precipitate was removed byThe enzyme sedimented as a single homogeneous centrifugation at 18,000 r.p.m. for 30 mm. at 4°component with a sedimentation coefficient of (Stage 3). The enzyme solution was concentrated 54°= 1.6X 10 (Fig. 2). Ultracentrifugal to a volume of 2 ml by ultrafiltration at 4° underanalysis of psoriatic RNase at Stage 4 of purifica- vacuum, using Visking cellulose casing. Thetion using a double sector cell with the buffer concentrated solution was dialyzed against adialysate as base line also revealed one homogene- 0.02 M Tris-HC1 buffer solution of pH 7.2 over-ous component of very low molecular weight. night in the cold. The dialyzed material was Starch Gel Electrophoresis. The limitation of centrifuged to remove fiocculent material. Theultracentrifugal analysis in determining homo- supernatant solution was brought to 0.8 am-geneity lies in its failure to reveal whether a monium sulfate saturation and allowed to standsingle component represents one molecular com- at 4° overnight. The material was then centri-ponent or several molecular components of the fuged at 4,000 r.p.m. for 20 mm. at 4°. The sedi-same molecular weight. For this reason, psoriatic ment was dissolved in 2 ml of 0.02 M Tris-HCIRNasc at Stage 4 of purification, i.e., before buffer of pH 7.2 containing 0.1 M NaC1 andDEAE treatment, was examined by starch gel dialyzed for four hours at 4° against this bufferelectrophoresis. Psoriatic RNase, although homo- solution (Stage 4). The dialyzed enzyme solutiongeneous on ultracentrifugal analysis, was found was mixed with 5 volumes of 0.05 M NaHCO3to contain several components on electrophoretic solution and applied to a DEAE column whichanalysis (Fig. 3). It contained two major com- had been equilibrated with a 0.05 M NaHCO1ponents migrating toward the cathode and solution. The enzyme was washed through theseveral small components migrating toward the column using a 0.05 M NaHCO3 solution asanode. Of the 2 components migrating toward eluent (Stage 5). The procedure yielded a RNasethe cathode one moved at a rate much faster than preparation which had been purified approxi-that of pancreatic RNase. This fast moving mately 25-fold (Table I). component subsequently was identified as psori- Ultracerttriju gal Analysis* of Psoriatic RNase.atic RNase. The sedimentation characteristics of psoriatic In view of the heterogeneous nature of the RNase at Stage 4 of purification were determinedenzyme preparation at Stage 4 of purification, *Wewish to thank Dr. Rudy H. Haschemeyerfurther purification was achieved using a 1 x 20 of the Biochemistry Department, Tufts IJniver-cm DEAE column. The elution pattern of the sity School of Medicine, for making the ultracentrifuge runs. protein passing through a DEAE column with a 532 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY

0.05 M NaIICO3 solution as eluent showed oneone of 88; and in tube 11, one of 29. Thus, ilNase major protein peak extending from tubes 8—11activity was highest in tube S and then decreased (Fig. 4). In tube 8 the RNase had a specificprogressively. The proteins eluted in the single activity of 177; in tube 9, one of 103; in tube 10,peak extending from tubes S to 11 consisted of the two protein components which had migrated toward the cathode on starch gel electrophoresis + (cf. Fig. 3), while the proteins which had migrated toward the anode were retained by the DEAE column. Psoriatic RNase was eluted first, followed by a second protein component of unknown nature which did not possess RNase activity. The material in tubes S through 11 obtained from the DEAE column was concentrated and resubmitted to starch gel electrophoresis. The material in tube 5, which contained psoriatic RNase of the highest specific activity, consisted of only one component which moved toward the cathode at a much faster rate than crystalline bovine pancreatic RNase (Fig. 5). The second protein component which migrated toward the cathode at a slower rate than psoriatic RNase FIG. 3. Starch gel electrophoresis of psoriaticwas eluted from the DEAE column after psoriatic RNase and crystalline bovine pancreatic RNaseRNase. The ratio of psoriatic RNase to this using the discontinuous borate buffer system,second component progressively decreased from pH 8.0. Sample 3 corresponds to crystalline bovine pancreatic RNase and sample 4 to psoriatictubes 9 through 11. RNase at Stage 4 of purification (cf. Table I). Substrate Specificity of Psoriatic RNase. After

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TUBE Fiu. 4. Elution pattern of psoriatic RNase which had been applied to a 1 X 20 cm DEAE column at Stage 4 of purification. The specific activities of psoriatic RNase for tubes 8 through 11 are in paren- theses. PSORIATIC RNASE 533

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Fin. 5. Starch gel electrophoresis of psoriatic 04 RNase in tubes 8, 9, 10, and 11 obtained after DEAE elution, and of crystalline bovine pancreatic RNase (p) using a discontinuous buffer 6.0 7.0 8.0 system, pH 8.0. PH Fin.6. pH dependency of psoriatic RNase. pH 5.0, 0.1 M sodium acetate buffer; pH 5.8 and 6.6, TABLE II 0.1 M sodium phosphate buffer; pH 7.4 and 8.0, Purine and Pyrimidine Composition of RNA Before 0.1 M Tris-HC1 buffer. and After Treatment with Psoriatic RNase The purine and pyrimidine composition of pH Dependency of Psoriatic RNase. The pH purified yeast RNA before and after prolongedoptimum of psoriatic RNase was approximately incubation with psoriatic RNase was determined6.6 (Fig. 6). on HC1O4 hydrolysates of the untreated RNA and Immunochemistry of Psoriatic RNase. Psoriatic the nondialyzable "core" RNA. RNase was found to be immunochemically

Composition of Non-Dialyzable distinct from crystalline bovine pancreatic RNase Treatment with RNA Purines since, on using the Ouchterlony agar double Psoriatic RNase Pyrimidines diffusion technic (18), it did not cross react with Ad. Gu. Ur. Cyt. antibody to pancreatic RNasef (Fig. 7). moles/iSO moles Before 21 30 22 27 1.0 DISCUSSION After 26 48 15 11 2.8 We have found ribonuclease in psoriatic scales in high concentration, at least 3 times greater than that found in normal human epidermis by prolonged incubation of yeast RNA with psoriaticSantoianni and Rothman (5). Therefore, RNA RNase there was in the resulting "core" RNAaccumulation does not result from a deficiency a decrease in the relative concentration of the twoof this enzyme. Our finding is in agreement pyrimidines (cytosine and uracil) and an increasewith the suggestion of Wheatley and Farber; (2) in the relative concentration of the two purinesthat the high concentration of RNA in psori- (adenine and guanine) (Table II). The ratio ofatic scales is the result of excessive formation purines to pyrimidines in the yeast RNA beforerather than of impaired breakdown. We did not treatment with psoriatic RNase was approxi-determine whether or not psoriatic scales con- mately 1 to 1, while after treatment the ratio wastain a physiological RNase inhibitor as Roth 2.8 to 1. This indicates that the substrate specific-has found in rat liver (19, 20). ity of psoriatic RNase is the same as that of Psoriatic RNase has a sedimentation coefficient of S4 =1.6x10—'.In comparison, bovine bovine pancreatic RNase, namely, hydrolysis f Pancreatic RNase antibody supplied by Dr. of the phosphodiester linkages involving theMayo Uziel of the Walker Biochemical Labora- pyrimidine moieties of the RNA molecule (17). tory, Massachusetts Eye and Ear Infirmary. 534 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 7. Agar double diffusion, using antibody to bovine pancreatic RNase in top center well, crystalline bovine pancreatic RNase in bottom right well, and purified psoriatic RNase in bottom left well. pancreatic RNase has an 2o= 1.88x 10 SUMMARY (21) and a molecular weight of 13,700 (22). On RNase was found to be present in high con- the basis of its sedimentation velocity, psoriaticcentration in psoriatic scales. Therefore, RNA RNase also can be assumed to have a very lowaccumulation does not result from a deficiency molecular weight in the order of 14,000. Psoriaticof this enzyme. RNase was also similar to bovine pancreatic The RNase present in psoriatic scales has a very RNase in its reaction specificity since it specifi-low molecular weight and is, like pancreatic cally hydrolyzed the phosphodiester linkagesRNase, specific for the phosphodiestcr linkages involving the pyrimidine moieties of the RNAinvolving the pyrimidine moieties of RNA. The molecule. However, electrophoretic analysisenzyme is a more basic protein than pancreatic indicated that it was a much more basic proteinRNase. than bovine pancreatic RNase and as might be REFERENCES expected, psoriatic RNase was immunochemically 1. STEIGLEnER, G. K.: Morphologische und histo- distinct from bovine pancreatic RNase. chemische Befunde in pathologischer Horn- schicht, insbesondere bei Parakeratose. Em We have shown that in addition to the high Beitrag zur Biologic der Hautobcrfiache. RNA and DNA content reported by Wheatley Arch. Klin Exp. Derm., 207: 209, 1958. and Farber (2), psoriatic scales contain a high 2. WHEATLEY, V. R. AND FARBER, E. M.: Chem- istry of psoriatic scales. J. Invest. Derm., RNase activity. Whereas, both RNA and DNA 36: 199, 1961. are degraded during normal epidermal keratiniza- 3. STEIGLEDER, C. K.: Zur Funktion der Akan- those. Arch. Klin. Exp. Derm., 200: 377, tion, they persist in psoriasis. It is noteworthy 1955. that although psoriatic scales contain large 4. FLEsCE, P. AND E50DA, E. C. J.: Pentoses in amounts of free uracil (2), we have found the horny layers, a simple color test for the diagnosis of psoriasis. J. Invest. Derm., 32: nucleotide present in lowest concentration in the 437, 1960. RNA of psoriatic scales was uridylic acid (23). 5. SANTOIANNI, P. AND ROTHMAN, S. Nucleic The functions of RNA as a reactant and of DNA acid-splitting enzymes in human epidermis and their possible role in keratinization. J. as the specificity determinant in protein bio- Invest. Derm., 37: 489, 1961. synthesis indicate that the anomalous protein 6. STEIGLEDER, C. K. AND RAAB, W. P.: The localization of ribonuclease and deoxyribo- synthesis in psoriasis probably results from a nuclease activities in normal and psoriatic disturbed nucleic acid metabolism. epidermis. J. Invest. Derm., 38: 209, 1962. PSORIATIC RNASE 535

7. CRESTFIELD, A. M., SMITH, K. C. AND ALLEN, 16. ALEXANDER, M., HEPPEL, L. A. AND Hua- F. W.: The preparation and characteriza- wTTz, J.: The purification and properties of tion of ribonucleic acids from yeast. J. Bin!. micrococcal nuclease. J. Biol. Chem., 236: Chem., 216: 185, 1055. 3014, 1961. 8. FIsKE, C. H. AND SUBBAROW, Y.: The colon- 17. SCHMIDT, 0.,CUBILEs,H., Z6LLNEH, N., metric determination of phosphorus. J. HECHT, L., STEICKLER, N., SERAIDAEIAN, Biol. Chem., 66: 375, 1025. K., SERAIDARIAN, M. AND THANNHATJSEE, 0. MCDONALD, M. R.: Ribonuclease, in S. P. S. J.: On the reaction or ribonuclease. J. Colowick and N. 0. Kaplan (Editors), Biol. Chem., 192: 715, 1951. Methods in Enzymology, Vol. II, p. 427.18. OUCHTEHLONY, 0.: Antigen-antibody reac- New York, Academic Press, Inc., 1955. tions in gels. IV. Types of reactions in co- 10. MACFADYEN, D. A.: The nuclease activity of ordinated systems of diffusion. Acta. Path. Bacillus subtilis. J. Biol. Chem., 107: 297, Microbiol. Scand., 32: 231, 1953. 1934. 19. ROTH, J. S.: Hibonuclease. VII. Partial purifi- 11. Lowny, 0. H., R0SEBE0UOH, H. J., FARa, cation and characterization of a ribonuclease A. L. AND RANDALL, R. J.: Protein measure- inhibitor in rat liver supernatant fraction. ment with the Folin phenol reagent. J. Biol. J. Biol. Chem., 231: 1085, 1958. Chem., 193: 265, 1951. 20. ROTH, J. S.: The distribution and some proper- 12. WARBUEG, 0. ANn CHEI5TIAN, W.: Isolierung ties of ribonucicase inhibitor. Fed. Proc., and Kristallisation des Garungsfermcnts 21: 380, 1962. Enolase. Biochem. Z., 310: 384, 1942. 21. SHAcHMAN, H. K.: IJltracentnifugation, diffu- 13. BENDIcH, A.: Methods of characterization of sion and viscometry, in S. P. Colowick and nucleic acids by base composition, in S. P. N. 0. Kaplan (Editors), Methods in Enzy- Colowick and N. 0. Kaplan (Editors), mology, Vol. IV, p. 32. New York, Aca- Methods in Enzymology, Vol. III, p. 715. demic Press, Inc., 1957. New York, Academic Press, Inc., 1957. 22. Hias, C. H. W., MOORE, S. AND STEIN, W. H.: 14. WYATT, 0. H.: The purine and pyrimidinc Peptides obtained by tryptic hydrolysis of composition of desoxypentose nucleic acids. performic acid-oxidized ribonuclease. J. Biochem. J., 48: 584, 1951. Biol. Chem., 219: 623, 1956. 15. BARRETT, H. J., FRIESEN, H. AND AsTwooD, 23. Liss, M. AND LEVEE, W. F.: The amino acid E. B.: Characterization of pituitary and and nuelcotide composition of the proteins peptide hormones by electrophoresis in and of RNA of psoriatic scales. J. Invest. starch gel. J. Biol. Chem., 237: 432, 1962. Derm., (in press). DISCUSSION DR. DAPHNE ANDERSON ROE, Ithaca, N. Y.: periments that maeromoleeular substances can I iery much enjoyed Dr. Liss's paper. I wouldact as RNase inhibitors. Our own recent studies like to ask him whether he has had any evidencehave supported this. that there is high ribonuelease inhibitor activity DR. MAURICE Liss, (in closing): In answer to in psoniatie epidermis? In actual practice, thereDr. Roe's question, it is known that there are is inhibition of this catabolic process. The factRNase inhibitors in other tissues. We have not that there are maeromoleeular substances in thedetermined whether or not any physiological psoriatic epidermis suggests a medium for ribonu-inhibitors of RNase are present in psoriatie elease inhibition. It is known from previous cx-scales.