Quantification of small amounts of hemoglobin in polyacrylamide gels with benzidine

ANALYTICAL

BIOCHEMISTRY

94,211-219

(1979)

Quantification of Small Amounts of Hemoglobin Polyacrylamide Gels with Benzidinel

in

ROBERT H. BROYLES,BRIAN M. PACK,STUART BERGER, ANDALLAN R. DORN Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center. Oklahoma City, Oklahoma 73190, and Department of Zoology, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53201

Received July 31, 1978 Benzidine-hydrogen peroxide is a sensitive blood stain. Since benzidine is carcinogenic, less hazardous substitutes have been sought which retain the specificity and sensitivity of benzidine. We have tested two benzidine derivatives, 3,3’-dimethoxybenzidine and 3,3’,5,5’tetramethylbenzidine, reported to be satisfactory substitutes, but have found both to be unsatisfactory for quantifying small amounts of hemoglobin (Hb) on polyacrylamide gels. However, our data show that benzidine stains gels with an intensity proportional to the amount of Hb present when the staining time and temperature are controlled. The stain is fast, sensitive, and specific for Hb, gives very little background stain, and is stable if the gels are thoroughly rinsed and stored in distilled-deionized water. The relative amounts of Hb in different bands or different gels may be quantified later by densitometry. Procedures are suggested for using benzidine while giving adequate attention to governing regulations and personnel safety.

In the course of our work on organspecific hemoglobin synthesis in bullfrog larvae (l-3), we have found it necessary to be able to detect and quantify in a relative way small amounts of hemoglobins separated by electrophoresis in polyacrylamide disc gels. Since it has been necessary to do such separations on extracts of cultured erythropoietic organs (1,3), we have required a stain which is sensitive and specific for hemoglobin. We have found benzidineH,O, is sufficiently sensitive and specific for this application. In this report we show that when used in the proper way the stain can be quantitative. Because benzidine is highly carcinogenic, we have sought a substitute which would ’ Supported in part by NIH Grant 5 ROl AM21386 from the National Institute of Arthritis, Metabolism and Digestive Diseases and by the Graduate School of the University of Wisconsin-Milwaukee. 211

meet the needs stated above. Although a variety of compounds have been tested by others for staining starch electrophoretograms (4) and for forensic and other applications (5,6), it appeared from the literature that only derivatives of benzidine were likely to have the desired specificity. We therefore tested 3,3’-dimethoxybenzidine and 3,3’,5,5’-tetramethylbenzidine, two derivatives of benzidine that have been reported to have the desired characteristics of sensitivity, specificity, and low carcinogenicity (4,6). As we report below, we have found neither substitute to be as desirable as benzidine for staining hemoglobins in polyacrylamide gels. In light of the desirability for the continued use of benzidine in this and related applications, we suggest some procedures for its use which will hopefully ensure the protection of laboratory workers and allow 0903-2697/79/05021 I-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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wg Hb

FIG. 1. Graph of total area under hemoglobin peaks on densitometer tracings of stained polyacrylamide gels versus micrograms of total hemoglobin electrophoresed. Electrophoresis of hemoglobins, staining of gels, and quantitative densitometry of stained hemoglobin bands were performed as described under Materials and Methods. Gels were stained either with 0.2% benzidine-hydrogen peroxide for 10 min at 25°C (0) or with 1% amido black for 45 min at room temperature (0).

adherence to regulations of benzidine .

governing

the use

METHODS

Chemicals. Benzidine base, benzidine di3,3’-dimethoxybenzidine hydrochloride,2 base (0-dianisidine), and 3,3’-dimethoxybenzidine hydrochloride were obtained in powder form from Sigma Chemical Company, St. Louis, Missouri. 3,3’,5,5’-Tetramethybenzidine powder was purchased from Tridom Chemical Inc., Hauppauge, New York. Equine skeletal muscle myoglobin, horseradish peroxidase, bovine liver catalase, and hematin were purchased from Sigma. Cytochrome &, was donated by Dr. 2 Benzidine base is no longer in the Sigma catalog, and benzidine dihydrochloride is available from Sigma only in the technical grade at 75% purity. We have found that impure benzidine works as described in this paper if the actual concentration with respect to benzidine is on the order of that stated here.

ET AL.

Shigemasa Osaki. Reagents used in polyacrylamide gel electrophoresis were also obtained from Sigma, with the exception of TEMED (N,N,iV’,N’-tetmethylmethylenediamine), which was obtained from Eastman Kodak Company, Rochester, New York. Polyacrylamide was recrystallized from chloroform before use (7). All other chemicals used were reagent grade. All solutions were made with water purified by a reverse osmosis-deionizer system (Culligan Aqua-Cleer 50, D-25) or by distillation followed by deionization. Electrophoresis. Hemoglobin solutions were prepared from blood of bullfrog (Rana caresbeiana) tadpoles as previously described (1,2). The concentrations of hemoglobin in solutions used for electrophoresis were determined spectrophotometrically after mixing the hemoglobin with Drabkin’s reagent; the procedure and reagents in Sigma Kit No. 525 (Sigma Chemical Co.) were used. Amounts of hemoglobin ranging from 0.86 to 55 pg were layered onto 7% polyacrylamide disc gels and electrophoresed using the system of Clarke (8) as we have previously described (l-3). No stacking gel was used since we have found that it does not improve the separation of hemoglobins. Staining solutions and procedures. For obtaining the data in Fig. 1 and Table 1, a stock solution of 0.2% benzidine dihydrochloride (w/v) in 1.0 M acetic acid was used. (The stock solution may be stored at 4°C for months.) Immediately before applying the stain to the gels, 0.2 ml of 30% H,Oe was added to 100 ml of the stock benzidine solution. After adding the HzOz, the staining solution was used within 2 h. (A stock solution with H202 added will turn brown when stored at 4°C overnight and will have a noticeable tint when stored frozen for a few days.) The staining solution was kept at 25°C in a controlled water bath before and during staining. Gels were stained in Pyrex test tubes for the times indicated in the legend

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to Fig. 1 and in Table 1. At the prescribed time, the staining solution was drained from each tube and immediately replaced by distilled-deionized water. The water was changed two times at approximately 20-min intervals, and the gels were allowed to sit in the last change of water overnight. The gels were then scanned with a densitometer and the amounts of hemoglobin in the stained bands determined as described below. It should be noted that stock solutions of benzidine dihydrochloride and 3,3’dimethoxybenzidine hydrochloride were made by dissolving the powders in 1 M acetic acid and making the solution up to volume with 1 M acetic acid. However, when the base form of either compound was used, the powder was first dissolved in an appropriate volume of undiluted acetic acid and then made up to volume with distilleddeionized water. Similar procedures were used in testing the two benzidine derivatives, 3,3’-dimethoxybenzidine and 3,3’,5,5’-tetramethylbenzidine. Stock solutions of 0.1% benzidine and of the two derivatives were also tested in a similar manner. Staining solutions of benzidine and the two derivatives were also prepared and tested with the procedure used by Owen et al. (4) for starch gels, in which benzidine or a derivative is dissolved in 70 ml ethanol and made up to 100 ml with acetate buffer (pH 4.7) and water. As with the other staining method, hydrogen peroxide was added immediately before staining. A general protein stain, amido black, was also used for comparison. A 1% stock solution (w/v) of amido black (Amid0 Black lOB, E. Merck, Darmstadt, Germany) was made in 7.5% (w/v) acetic acid and filtered before use. The stain was applied directly to gels in Pyrex test tubes at room temperature, usually for 1 h. After staining, the excess amido black was washed out of the gels over a 48-h period by repeatedly soaking the gels in 7.5% acetic acid. The destained gels were stored in 7.5% acetic acid, and hemoglobin

TABLE STAINING

TIMES

PRODUCING

BETWEEN PEAK OF HEMOGLOBIN

Stain” Benzidine-H,O,

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GELS

(0.2%)

Amido black (1%)

1 LINEAR

AREAS AND APPLIED

RELATIONSHIPS AMOUNTS TO GELS

Staining time (min)

Linear range* (ba hemoglobin)

5-9 lo- 12 19-24 30-60

3.4-55 1.7-28 0.8-14 1.7-14

a Stains were prepared and applied to polyacrylamide gels containing hemoglobins separated by electrophoresis as described under Materials and Methods. * Linear portions of graphs of areas under total hemoglobin peaks versus micrograms of hemoglobin mixture electrophoresed were obtained as described in the legend to Fig. 1 and under Materials and Methods.

bands on the gels were quantified as described below for benzidine-stained gels. Quantification of hemoglobins in gels. Benzidine-stained gels which had been stored in distilled-deionized water overnight and destained amido black-stained gels were scanned with a GCAlMcPherson Model EU 721 ratio-recording spectrophotometer with an EU 705-11 gel scanner attachment. The gels were scanned at a rate of 5 mmlmin with a 0.05-mm slit at a wavelength of 540 nm. An attached chart recorder was set at a speed (usually 17.5 mm/ min) which spread the peaks out. The optical density range was always set so that the highest peak on the tracing occupied as much of the lo-in. span of the recorder paper as possible. The combination of a slow scanning speed for the gel drive, a small scanning slit, and a chart record that was expanded in both axes allowed the best possible quantification of the areas under the peaks which corresponded to hemoglobin bands on the gels. Densitometer tracings have also been made with an ISCO Model 1310 gel scanner coupled to an ISCO Model UA-5 absorbance monitor.

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Areas under hemoglobin peaks on the densitometer tracings were determined either one of two ways: (i) Peaks were cut and weighed on a five-place analytical balance; or (ii) the areas under the peaks were determined with a compensating polar planimeter. Areas determined by both methods for a single tracing showed that there was good agreement between the two methods. Precautions taken in using benzidine. Since the observations described in this paper were made over a long period of time, our precautions in using benzidine have increased with our knowledge of its dangers and with the increasingly stringent regulations that have come to govern its use (9). Most of the experiments were done at a time before OSHA regulations (9) were applied to the academic laboratories in which we were working. For most of the experiments described, the compounds were carefully weighed in a low-draft area by an investigator wearing full-length protective clothing with overlapping gloves and a respiratory mask which filters out both particulates and organic vapors. The solids were then dissolved in the indicated solvents in a fume hood by the same investigator wearing the same protective garb. Once the stock solutions had been made, they were stored in bottles with warning labels in a refrigerator which also contained a warning on the door. Investigators using the stock solutions were also required to wear protective clothing and gloves that overlapped the sleeves, and the staining solutions were applied to the gels in a fume hood. Used staining solutions were recovered and stored in closed marked containers until sufficient volumes were accumulated for disposal. The used staining solution was then mixed with diesel oil and incinerated at high temperatures. The OSHA regulations (9) that now apply to many academic laboratories are much more stringent than the precautions indicated above, when concentrations of benzidine of 0.1% (w/v) or greater are used. For that reason, we now use benzidine at a

ET AL.

concentration of slightly less than 0.1%; but the precautions we have used in the past are still enforced. Further recommendations regarding the use of benzidine are given under Discussion. RESULTS

Quantification of Hemoglobins Polyacrylamide Gels with Benzidine-H,Oz Staining

in

Various amounts of a red blood cell lysate from bullfrog tadpoles, which contained the four larval hemoglobins of this species, were electrophoresed on different polyacrylamide gels and the gels were stained with either 0.2% benzidine dihydrochloride-hydrogen peroxide or amido black. Densitometer tracings were made for all gels, and the areas under the peaks corresponding to hemoglobins were determined as described under Materials and Methods. In Fig. 1, the results of one such experiment are presented as a graph of total area (cm2) under the four hemoglobin bands versus the amount (pg) of total hemoglobin applied to the gel for both benzidine-H,O, stained gels and gels stained with amido black. It can be seen that the relationship between the area under the hemoglobin peaks on the densitometer tracings and the amount of hemoglobin applied to the gels is linear from 1.7 to 14 pg of total hemoglobin per gel, for both types of stain. Gels were also layered with 0.8 and 0.4 pg of total hemoglobin, and bands could be seen on gels stained with benzidine (data not shown). However, the peaks on densitometer tracings of gels with these lower amounts of hemoglobin were so small that precise determinations of the areas under such peaks could not be made. As indicated in Fig. 1, the benzidine stain was found to give a more intense stain than amido black for the range of hemoglobin concentrations tested. The relationship between the area under an individual hemoglobin peak and the amount of total hemoglobin applied to a gel

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was also found to be linear for each of three tadpole hemoglobins when the gels were stained with benzidine-H,O,, and it was found that as little as 0.4 pg of an individual hemoglobin may be quantified on these gels (data not shown). Thus, the technique can also be used to quantify small amounts of individual hemoglobins separated from a mixture. The gels used to obtain the data in Fig. 1 were stained for 10 min at 25°C. As indicated in Table 1, the linear range for staining with benzidine-H,O, may be shifted in either direction by altering the staining time. However, we have observed that a single hemoglobin present in an amount less than 0.1 pg on a gel is not likely to be quantifiable, even when the staining time is increased to 30 min or more. We have observed that, for the amounts of hemoglobin indicated in Table 1, little if any additional staining occurs after about 20 min; gels containing identical amounts of hemoglobin and stained either 19 or 24 min stained to almost exactly the same degree (data not shown). We have found that, for gels containing 28 ,ug or less total hemoglobin, the degree of staining is linear with time between 6 and 12 min (data not shown). In testing different lots of benzidine and benzidine dihydrochloride obtained from the same sunplier, we have noted variations in both the appearance of the powder (color ranged from white to light brown to violet) and the staining intensity with different amounts of hemoglobin. It is necessary to construct a curve like that in Fig. 1 for each lot of benzidine, if absolute rather than relative quantitation of hemoglobins in the gels is desired. Tests with Benzidine

Derivatives

We compared the staining characteristics of benzidine with that of 3,3’-dimethoxybenzidine (0-dianisidine) and 3,3’,5,5’tetramethylbenzidine at two concentrations, 0.2 and 0. I%, in 1 M acetic acid, and at 0.1% concentration in 70% ethanol-O.15 M

GELS

215

sodium acetate buffer, pH 4.7, as used by Owen et al. (4) for starch gels. We have found that solutions containing ethanol or methanol are unsatisfactory for staining polyacrylamide gels. The gels stained poorly or not at all, and the gels shrank to varying degrees in the presence of the alcohols. When benzidine dihydrochloride, 3,3’dimethoxybenzidine hydrochloride, or 3,3’ ,5,5’-tetramethylbenzidine was delivered to the gels in 1 M acetic acid at concentrations of 0.1 and 0.2%, all of the hemoglobins were stained. However, the degree of staining, time required for staining, stability of the stained bands, and amount of background staining on the gels varied with the compound used and its concentration. 3,3’-Dimethoxybenzidine was found to be less sensitive than benzidine at both 0.1 and 0.2% concentrations and required longer staining times to give detectable hemoglobin bands at some hemoglobin concentrations (data not shown). At a concentration of 0. l%, dimethoxybenzidine applied for 45 min gave a less intense stain than 0.1% benzidine applied for 5 min. The hemoglobin bands stained with dimethoxybenzidine were more diffuse than those stained with benzidine, and there was a significant amount of brown background stain (the hemoglobin bands were also stained brown) in other parts of the gel that could not be washed out with either acetic acid or water. Gels stained with either concentration of benzidine for 5 or 10 min had almost no detectable background stain. 3,3’,5,5’-Tetramethylbenzidine was found to be a very sensitive stain and to stain gels quickly at both concentrations in 1 M acetic acid. The hemoglobin bands stained a bright green-blue. However, the rest of the gels also had a marked green-blue color; there was much more background staining than found with either benzidine or dimethoxybenzidine. The stained hemoglobin bands began to diffuse immediately, continued to broaden with time, and were not stable

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enough to allow even relative quantitation. When the gels stained with tetramethylbenzidine were stored in either water, 1 M acetic acid, or higher concentrations of acetic acid, the bands continued to diffuse with time and disappeared overnight. Specificity

of the Different

Stains

We have previously established by several means that the benzidine-stained bands we observe on polyacrylamide gels of electrophoresed extracts of erythropoietic organs are authentic, known larval hemoglobins of Rana catesbeiana (1). Part of our evidence on this point, which has not been published before, included a study of the electrophoresis and staining characteristics of known heme-containing proteins and hematin. The results of these experiments are briefly summarized below. Catalase, horseradish peroxidase, and hematin all exhibit peroxidase activity and a stain on gels with the benzidine reaction. However, catalase stains with only about 1/20th the intensity of hemoglobins (50 pg of catalase gives only half as much stain as 5 pg of hemoglobin). Likewise, horseradish peroxidase stains with only about l/lOOth the intensity of hemoglobins, and the initial stain with benzidine is of a different hue (blue-green versus bright blue for hemoglobin). None of these proteins is likely to cause any interference with detection of hemoglobins in an electrophoretic system such as ours, since their migrations are vastly different: Peroxidase migrated near the top of the gel while the hemoglobins all migrated in the bottom one-third; catalase is a very large protein and barely enters the gel at all (it is greatly retarded by the molecular sieving effect of 7% polyacrylamide gels); and hematin migrates with the electrophoretic front (the bromophenol blue tracking dye). Cytochrome b5 migrated in the hemoglobin region of the gel, but even a large amount (about 50 pg) was barely detectable with the benzidine stain. Myoglobin stains very

ET

AL.

weakly with benzidine; 50 pg gave a stained band that was barely detectable. In contrast to weak staining by benzidine, we found that catalase stained much more strongly with 3,3’-dimethoxybenzidine, possibly indicating that this benzidine derivative is not as specific for hemoglobin. For this application, we suspect that 3,3’,5,5’-tetramethylbenzidine is also not specific for hemoglobin. We found that the stock solution of this compound in 1 M acetic acid (the only solvent that seemed suitable for our application) began turning greenblue as soon as hydrogen peroxide was added. In a matter of minutes, before the stain could be applied to gels, the solution had turned a dark green-blue. As mentioned above, gels stained with this compound exhibited a large amount of background stain. These observations indicated to us that the compound is easily oxidized in the presence of hydrogen peroxide and that it probably would not have the desired specificity. This point was not pursued further since the compound was found to be unsatisfactory for another reason, i.e., the stained bands diffused too rapidly to allow even semiquantitative evaluations to be made. Tests with 0.1% Benzidine Dihydrochloride Since the OSHA regulations (9) do not pertain to the use of benzidine in concentrations below 0. I%, we have tested the staining properties of 0.1% benzidine dihydrochloride in 1 M acetic acid. (Since the dihydrochloride form was used, the actual concentration with respect to benzidine was 0.072%.) We have found that about 30% of the sensitivity is lost with the lower concentration of benzidine, when gels containing the same amounts of hemoglobin are stained for the same length of time as those with 0.2% benzidine dihydrochloride. Some but not all of the loss in sensitivity was regained by staining longer since, as found when 0.2% benzidine dihydrochloride was

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GELS

217

as a carcinogen, although Ferretti and coworkers (11) have found benzidine, Odianisidine, and 0-tolidine to be mutagenic in the Ames assay (12), and Collier (13) has cautioned laboratory workers about the use of these compounds. 0-Dianisidine has been reported to be four times as sensitive as benzidine for staining hemoglobins in starch gels and to give a color which is stable for at least a month (4). Tetramethylbenzidine has also been reported to be four times as sensitive as benzidine in the detection of blood, to be specific and easy to use, and to have low or no carcinogenic activity in DISCUSSION rats (6). Although benzidine has been used for We have found both of the above commany years to detect small amounts of blood pounds to be much less satisfactory than or hemoglobin in various types of samples benzidine for this application (see Results). (4-6), it has not been routinely used as a 0-Dianisidine was found to be less sensitive quantitative stain for hemoglobins on gels and less specific than benzidine, to require because of uncontrolled variations in the de- longer staining times, and to give more backgree of staining. We have found that if the ground color to the gels and more diffuse staining time and temperature are controlled, hemoglobin bands, when applied to polybenzidine can be used to quantify small acrylamide gels. Although it was very sensiamounts of hemoglobins separated by polytive and stained rapidly, tetramethylbenziacrylamide disc gel electrophoresis. As dine was found to be entirely unsatisfactory shown by the data in Fig. 1 and Table 1, because it gave a large amount of background under controlled conditions the intensity of stain and the stained bands were not stable the stain is directly proportional to the and began diffusing in a matter of minutes. amount of hemoglobin electrophoresed over Because of the large amount of background a lo- to 20-fold range. As indicated in Table 1 stain on the gels and the observation that and under Results, the benzidine stain was the staining solution turned a dark greenfound to be rapid, sensitive, specific for blue as soon as hydrogen peroxide was hemoglobin, to give very little background added, we suspect that tetramethylbenzidine stain in the gels, and to give stained bands does not have the desired specificity in this which are stable in water and can be quantiapplication. We did not test compounds fied at a later, convenient time by densi- such as 0-tolidine, 0-anisidine, diphenyltometry, when the stain is used as described amine, guaiacol, and 0-toluidine because under Materials and Methods. 0-tolidine was also found to be mutagenic Although benzidine has the advantages in the Ames assay (11) and the other comnamed above, it has the disadvantage of be- pounds are known not to be as specific as ing carcinogenic (6,ll). We, therefore, tested benzidine . two derivatives of benzidine that have been Holland et al. (6) have noted that orthoproposed as suitable substitutes for benzidine hydroxylation of benzidine and other aroin most apphcations: 3,3’-dimethoxybenmatic amines, presumably by the liver, is an zidine (4) and 3,3’,5,5’-tetramethylbenzidine important metabolic step in the carcinogenic (6). 0-Dianisidine (3,3’-dimethoxybenziaction of these compounds. Therefore, they dine) has not yet been formally classified prepared and tested as a substitute the ben-

used, very little additional staining occurred after 20 min. However, we have found that the sensitivity of the stain with either concentration of benzidine can be increased 10 to 20% by converting the hemoglobin to the cyanomethemoglobin form with Drabkin’s reagent (10) before electrophoresis. The advantages of rapid staining, specificity for hemoglobin, low background staining, stability of stained bands, and quantifiability are retained with the lower concentration of benzidine.

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zidine derivative 3,3’,5,5’-tetramethylbenzidine, in which or&o-hydroxylation should be impossible without some other, prior metabolic alteration of the compound. Although such a change in the molecule apparently did lower or possibly even eliminate its carcinogenicity, the four substituent methyl groups also apparently changed the behavior of the molecule in other ways. For our results indicate that, in aqueous mixtures, the colored product of tetramethylbenzidine formed by peroxidase activity of methemoglobin diffuses from the polyacrylamide gels as it is formed. Such is not the case with benzidine. As cited by Holland et al. (6), Duijn (1955) presented a scheme for the formation of colored products from the action of peroxidase/hydrogen peroxide on benzidine. The scheme leads to the formation of a polymer of the reaction products of benzidine. We postulate that the polymer of colored product formed in the benzidine reaction becomes trapped in polyacrylamide gels, giving rise to discrete, stable bands corresponding to the locations of the hemoglobins in the gel. We further postulate that 3,3’,5,5’-tetramethylbenzidine does not form such a polymer, or that the extent and rate at which a similar polymer might form is not sufficient to allow the colored product to be trapped in the gel. Apparently, the tendency of the benzidine molecule to behave in this way has been altered by the presence of the four methyl groups. The desired attributes for a benzidine derivative of low carcinogenic potential and ability to form discrete, stable bands in polyacrylamide gels may be mutually exclusive. For these reasons we have continued to use benzidine, but with great care. Because compliance with the OSHA regulations (9) governing the use of concentrations of benzidine of 0.1% (by weight or volume) or higher is impossible for our laboratory, as it might be for others (14), we now use benzidine in a concentration that is below 0.1% (w/v). We have found that 0.1% benzidine

ET AL.

dihydrochloride (or 0.07% benzidine base) is as satisfactory as 0.2% benzidine in every way except that a small degree of sensitivity is lost (see Results). Benzidine at the lower concentration is preferable to either of the derivatives tested for this application. However, we are of the opinion that 0.07% benzidine is not significantly safer than 0.2% and rigorously exercise the precautions outlined under Materials and Methods for storing and using solutions of benzidine. The problem of dealing with the solid form of benzidine in order to obtain a dilute solution remains. Once a bottle of benzidine powder is opened in a laboratory, the onerous provisions of the OSHA regulations must be met. A simple remedy would be to prepare a dilute solution of benzidine in a nearby laboratory which meets the OSHA regulations. The benzidine could then be transported to the laboratory in which it will be used. ACKNOWLEDGMENT We thank animals from

Mr. Gintaris Dargis for caring for the which the hemoglobins were obtained.

REFERENCES R. H., and Frieden, E. (1973) Narure New 1. Broyles, Biol. 241, 207-209. 2. Deutsch, M. J., and Broyles, R. H. (1975)DeveIop. Biol. 46, 227-231. 3. Broyles, R. H., and Deutsch, M. J. (1975) Science 190,471-473. 4. Owen, J. A., Silberman, H. J., and Got, C. (1958) Nature (London) 182, 1373. 5. Markarem, A. (1974) in Clinical Chemistry, Erinciples and Technics (Henry, R. J., Cannon, D. C., and Winkelman, J. W., eds.), pp. llll1214, Harper & Row, New York. 6. Holland, V. R., Saunders, B. C., Rose, F. L., and Walpole, A. L. (1974) Tetrahedron 30, 3299-3302. 7. Loening, U. E. (1967) Biochem. J. 102, 251-257. 8. Clarke, J. T. (1964) Ann. N. Y. Acad. Sci. 121, 428-436. 9. General Industry OSHA Safety and Health Standards (29 CFR 1910), OSHA 2206 (revised Jan-

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uary, 1976), Section 1910.1010, Benzidine, pp. 550-555, U. S. Department of Labor Occupational Safety and Health Administration. 10. Moss, B., and Ingram, V. M. (1%8) J. Mol. Biol. 32, 481-492. 11. Ferretti, J. J., Lu, W., and Liu, M. B. (1977)Amer. .I. Clin.

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12. Ames, B. N., Durston, W. E., Yamasaki, E., and Lee, F. D. (1973) Proc. Nat. Acad. Sci. USA 70, 2281-2285. 13. Collier, H. B. (1974) Clin. Biochem. 7, 3-4 (editorial). 14. Eckardt, R. E. (1974) Bull. N. Y. Acad. Med. 50, 620-625 (essay).