ANALYTICAL

96, 464-468

BIOCHEMISTRY

A Positive ROBERT Department

C. KARN, of Medical

(1979)

Zymogram MARGARET

Genetics,

Method

CRISP, EMILY

for Ribonuclease A. YOUNT,

Indiana University School of Medicine, Indianapolis, Indiana 46223

AND M. E. HODES II00

West Michigan

Street,

Received November 14, 1978 We have developed a rapid, sensitive, and specific zymogram for detecting ribonuclease (RNase). The method makes use of an agarose gel containing the small substrate UpA [uridylyl (3’ + 5’)-adenosine]. UpA is hydrolyzed by RNase to adenosine, which is deaminated by adenosine deaminase. The inosine so formed is linked by a series of enzymatic reactions (nucleoside phosphorylase, xanthine oxidase) to formation of a blue tetrazolium salt. This method is superior in that it entails a staining reaction only at sites of RNase activity (positive zymogram) rather than clearing of a background of RNA (negative zymogram), a process which is often mimicked by protein devoid of RNase activity.

Lack of a rapid, reliable and sensitive zymogram has been a major limitation to genetic studies of ribonuclease (RNase, ribonucleate 3’-pyrimidino-oligo-nucleotidohydrolase, EC 3.1.4.22). The RNase zymograms commonly used are negativestaining procedures. (In this report the term “negative-staining procedure” is defined as a zymogram technique in which the substrate is stained and the term “positivestaining procedure” is defined as a zymogram in which the product is stained.) In the negative-staining procedures, RNase activity is represented by clear areas on a background of stained (1-4) or acid-precipitated (5) RNA. These methods have several limitations. Diffusion of the patterns is promoted by the large amounts of enzyme and long periods of time required, and high concentrations of protein may cause clearing of the gel and a false positive reading. We have developed a new zymogram method which uses a dinucleoside monophosphate as substrate for RNase. This technique couples the RNase-catalyzed release of adenosine to a previously described (6) zymogram for adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). The resultant blue formazan indi0003-2697/79/100464-05$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

cates the site to which RNase has migrated. The advantages and limitations of this zymogram method are the subjects of this report. MATERIALS

AND METHODS

Materials. Dinucleoside monophosphates; adenosine deaminase, Type III; agarose, phenazine methosulfate; and MTT’ were purchased from Sigma Chemical Company. Nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) and xanthine oxidase (xanthine: oxygen oxidoreductase; EC 1.2.3.2) were from Boehringer/Mannheim, purified pancreatic bovine RNase from Miles Laboratories, and RNA (A grade) from Calbiochem. Cellogel sheets were purchased from Kalex Scientific Company. Biological samples. Human granulocytes were isolated by a modification of the method of Beutler et al. (7). Urine was lyophilized, reconstituted in one-fortieth volume of distilled water, and stored at -20°C. Human ’ Abbreviations used: UpA, uridylyl (3’ 4 5’). adenosine; ApU, adenylyl (3’ + 5’)~uridine; ApA, adenylyl(3’ --$ 5’) adenosine; ADA, adenosine deaminase; MTT, 3-(4,5-dimethyl thiazolyl-2)-2,Sdiphenyl tetrazolium bromide (thiazolyl blue). 464

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for the RNase-catalyzed production of adenosine. UpA gave a weak-staining reacREAGENTS FOR THE RNase POSITIVE STAIN tion when used as the sole substrate in the Volume zymogram mixture, while ApA and ApU Zymogram reagent w gave no reaction at all when each was used alone. The most intensive staining resulted 50 UpA + ApA (10 mg/ml in distilled water) from combination of UpA and ApA. By 200 Phenazine methosulfate (5 mglml) contrast, combining UpA and ApU gave a 200 MTT (5 mg/ml) 10 Nucleoside phosphorylase weak-staining reaction, only slightly stronger 10 Xanthine oxidase than that obtained with UpA alone, and 20 Adenosine deaminase combination of ApU and ApA gave no staining reaction at all. spleen from autopsy was extracted as deFigure 1 shows the complete zymogram stain obtained using UpA and ApA in conscribed previously (8). cert. A second gel, also shown in Fig. 1, RNase assay. Total activity against RNA was determined by the acid-soluble frag- illustrates the effect of omitting adenosine ments method (9). A unit of activity causes deaminase during the staining reaction. The lack of staining in the absence of adenosine the release of 1.0 Az6,, unit of acid-soluble deaminase demonstrates the dependence oligonucleotide in 1 min at 37°C. Cellogel efectrophoresis. Cellogel strips of the zymogram on the production of adenowere presoaked in running buffer (0.04 M sine. Also shown in Fig. 1, for comparative Na-barbital buffer, pH 8.6), samples of purposes, is a negative stain resulting from l-5 ~1 applied to the blotted strips and acidification of an RNA-impregnated overlay. electrophoresis at constant voltage of 250 V The positive zymogram technique has for 3-4 h performed as described (2). been used to stain commercial bovine RNase Negative-staining procedure. After elec- and the RNases found in human granulotrophoresis the Cellogel strips were applied cytes, spleen, and urine (Fig. 1). The detecto RNA-impregnated agarose gel (5). When tion of the commercial RNase and compariincubation was complete the undigested son of the patterns obtained with the positive RNA background was visualized by acidi- and negative staining methods indicate that fying the gel (5). it is actually RNase that is being stained Positive-staining procedure. Cellogel by the positive zymogram method. Also, strips were applied to an agarose gel con- cellulose acetate sheets were cut, following taining the positive zymogram reagents electrophoresis of RNase, and the strips (Table 1). The gel was prepared by dissolving eluted and activity was determined by the 0.1 g of agarose in 9.5 ml of boiling Soren- quantitative RNase assay (9) in order to sen’s phosphate buffer, pH 7.5 (10). The ascertain the direction and extent of migraagarose solution was cooled to 45°C before tion of RNase on the sheets. Figure 2 shows addition of the reagents listed in Table 1. the results of this experiment when perThe staining solution was poured onto a formed using granulocyte extracts. It is glass plate and allowed to gel in the dark. clear that the direction and extent of migraCellogel strips were held in contact with tion of RNase activity, as determined by the zymogram gel in a humid chamber in the quantitative assay method, is the same the dark for 1 h at 37°C. as that obtained by both the positive and negative zymogram methods. RESULTS Comparison of the degree of staining of Three dinucleoside phosphates, UpA, known amounts of commercial RNase ApA, and ApU, were tested as substrates stained by both the positive and negative TABLE

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FIG. 1. Comparison of RNase zymogram methods. (A) An RNA-impregnated gel visualized by acidification (negative zymogram). Cleared areas indicate RNase digestion of the substrate. Channel 1: commercial bovine pancreatic RNase; channel 2: human granulocyte RNase. (B) The positive zymogram method linking production of adenosine from UpA to an adenosine deaminase-nucleoside phosphorylase-xanthine oxidase reaction series. Channel 1: commercial bovine pancreatic RNase; channel 2: human granulocyte RNase; channel 3: human urine RNase; channel 4: human spleen RNase. (C) same as (B) except adenosine deaminase was omitted from the reaction series.

zymogram methods shows that the positive method is more sensitive by a factor of about 200. The new method was capable of detecting as little as 0.066 unit of RNase activity of the commercial preparation while the older method was capable of detecting 13.0 units of activity from the same commercial preparation. Cellulose acetate strips were stained over the pH range 6.0-9.0. The zymogram staining reaction occurs optimally between pH 7.0 and 7.5. Only very weak staining was seen below pH 7.0, and the zymogram background became too dark for distinct reading of the zymogram above pH 7.5. Human spleen extracts, granulocyte extracts, serum, and urine were subjected to electrophoresis on cellulose acetate sheets and stained for RNase activity. RNase activity from human spleen, granulocytes, and urine was stained by the zymogram method as shown in Fig. 1. Serum RNase was not stained. Spleen,

urine, and granulocyte RNases are within the pH range of the zymogram technique; whereas, serum RNase has its optimum activity above the pH range of the zymogram. DISCUSSION

The positive RNase zymogram method reported here has several advantages over the negative zymograms commonly used to detect RNase on electrophoresis media. The positive method is capable of detecting smaller amounts of RNase making it particularly useful for screening small samples of biological tissues and fluids such as those obtained for genetic screening studies. The staining reaction occurs more rapidly so there is less diffusion of the enzyme reaction products during staining and more distinct zones of enzyme activity. Several reports (1,3,4, I 1) have indicated that proteins, such as albumin, may pene-

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FIG. 2. Evidence for identity of zymogram-detected RNase activity and RNase activity eluted from a Cellogel strip following electrophoresis. Inset (A) shows a direct comparison of the positive (channel 1A) and negative (channel 2A) zymogram methods. Inset (B) shows a zymogram from the same Cellogel strip as the elution profile (channel 1B) and a strip of the gel stained for protein (channel 2B).

trate the RNA-impregnated negative staining overlay and give a false RNase reaction when the gel is acidified or stained for RNA. This has not been observed, nor would it be expected to occur, in the positive zymogram method since this method relies on the production of adenosine for the staining reaction to occur. The major limitation to the positive zymogram method is the relatively narrow pH range (7.0-7.5) in which staining occurs. The limiting factor is the pH dependence of adenosine deaminase. While this presents problems in detecting RNases active at high or low pH, the method is clearly useful in studies of RNases active around neutrality, such as the human spleen, granulocyte and urine RNases, and bovine pancreatic RNase. The action of RNase on UpA is enhanced by the presence of ApA. The RNase-cata-

lyzed hydrolysis of cyclic 2’,3’-uridylic acid at pH 7 is known to be activated by ApA (12). We are presently elucidating the role of ApA in the zymogram procedure. ACKNOWLEDGMENTS We would like to thank John M. Thomas for helpful discussions and Daniel Rudzinsky and Kathleen O’Connell for technical assistance. This is publication No. 78-44 and was supported in part by the Indiana University Human Genetics Center, PHS 50 GM 21054. R.C.K. was supported by PHS Career Development Award 1 KO4 AM 00284-01 and E.A.Y. was supported by PHS Training Grant TO1 GM 1056.

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9. Hodes, M. E., Yip, C. C., and Santos, F. R. (1967) Enzymologia 32, 241-255. 10. Sorenson, S. P. L. (1912) Ergeb. Physiol. 12, 393-532. 11. Ressler, N., Olivero, E., Thompson, G. R., and Joseph, R. R. (1966) Nature (London) 210, 695-698. 12. Wieker, H.-J., and Witzel, H., (1967) Eur. J. Biochem. 1, 251-258.