N e w Direct Fluorometric Method for Measuring Dehydrogenase Activity SIR: Spectrophotometry is generally preferred in enzyme assay over manometric and pH procedures, because of its hiplicity, its rapidity, and the capability of measuring lower enzyme and substrate concentrations. On the other harid, nmiy of the compounds utilized in spectrophotometric assays have tiecn observed to fluoresce, and since fluorescence procedures are several order> of inapnitude more sensitive than coloi~iiiietricmethods i6), assays based on flucrometry have reillaced its sister method in numerous cases (10). In recc'nt iiut~lications,WF' have described the 1)rel)aration of various esters of fluorescein (2,4 ) and resorufin ( 5 ) for use in the determination of very small quantities of lipase and cholinesterase,. respcctivelj-. I,on.ry and coworkers (?: 8) have descrilietl an assay procedure for dehydrogenases based 011 the fluorescence of oxidized S A D (nicotinamide adenine diiiurleotide) in basic solution. The assay of S A D + required heating the reaction products with 6 S sodium hydroxide for 5 to 30 minutes after ivhirh time the fluorescence of oxidized S h l ) was ~ read at an excitation wavelength of 360 nip and an eiiiission warelength of 460 nip (8). In a similar manner, the activity of malic acid dehydrogenase ( 7 ) was assayed. Becauhe of the time arid manipulation iiivolved in such l)rocedures, it seeined desirable to devel011a fluorescence assay Iirocedurc. in which a direct nieasurenient of the rate of fluorescence product ion in an enzyine reaction would be related to the activity of dehydrogcxriaseh. Preliniinar- exlierinients revealed the possibility of determining such enzymes by using resazurin in conjunction with the S.\ln+-SXDH system. The nonfluorescent material, resazurin (I) is converted to the highly fluorescent compound, resorufin (II), as follow :
pyruvate
NADH
f
H i 4-
Dependence of AF/At upon the Concentrations of LAD, Diaphorase, and Resazurin [Diaphorase], s F / i t , fluorescence [LAD], unit/ml. [Resazurin], Jf uni t/rnl. units per minute 0 080 6 x 0 015 0 000470 6 x 0 080 0 0T5 0 0023j 6 x 0 080 O 1.50 0 00470 6 X 0 080 0 730 0 0235 6 x 0 080 1 49 0 0470 0 080 6 x 1 0 F 14 3 0 470 0 080 6 x lo-' 1 LO 0 470 0 080 1 . 5 x 10-7 O 30 0 470 0 080 3 x 10-6 0 060 0 470 0 040 6 x 7 15 0 470 6 x 0 020 3 60 0 470 0 0080 6 x 1 42 0 470 0 0040 6 x 0 714 0 470 6 x 10-6 0 00040 0 0716 0 470
Table I.
The rate of change in the fluorescence of the solution with time, AF AtJ I$ proportional to the amount of lactic acid dehydrogenase (LAID)present in the solution. EXPERIMENTAL
Reagents. ENZYMES. Lactic acid dehydrogenase, LAD, 142 units per mg. (Calbiochemical Co., Los Angeles, Calif.). One unit of activity is that which causes an initial rate of oxidation of 1 pniole of XADH per minute under specified conditions at 25' C. ( I ) . Diaphorase (Korthington Xochemical Co., Freehold, S . J.), act,ivity 10.4 units per nig. One unit equals a decrease in absorbance of 1.00 per minute a t 600 mp using the dye 2,6-dichloroindophenol and the coenzyme S A I D H (9).
.\lcohol dehydrogenase, hDH (Wort,hington Biochemical Co.), activity 301 units per ing, One unit equals 1 pniole of S.4D+ reduced 1)er minute at 25" C. under the conditions specified ( 3 ) . SUBSTRATES. Resazurin (Eastnian Organics Co., Rochester, S . Y.). A stock 10-3.11 solution was prepared in methyl Cellosolve. Kicotinamide adenine dinucleotide, S*\D+ (Calbiochemical Co., 86.37c pure). h stock 10-3.11 solut'ion was prepared in triply dist,illed water.
+ NADH + H +
'a,zD
0-
Diaphorase
=
560 m p
Xeni = 580 mp
NADH
+ D P S H + H+
+ H - + resazurin ------+ diapilorase XXD+
6 Xex
+
CHBCH~OH S = \ D +;IDH CHsCHO
I
I
Apparatus. -111 fluorescence measurements were made with a n -AniincoBowman spectrophotofluoronieter, as described in a previous publication ( 6 ) . Procedure. D ET E RMI s A T I o N o F LAID. Two milliliters of 0.01.11 sodium lactate in 'Tris buffer, pH 9.0, 0.1 nil. of 10-3.11 S-AD', 0.1 nil. of 2 x 10-"11 resazurin, and 1 nil. of diaphorase (0.08 unit per 1111.) are placed in a 3.0-nil. fluoreveiicr cell thermostated at 25' C'. in an . h i n c o Bo\vnian sl,ectrol,hotofluoroiiictrr and the instrument is adjuhted to read zero fluorescence. AItzero time, 0.1 nil. of a solution of the L.11) (containing 0.00040 to 0.50 unit I)rr nil.) is then added. and the change in the fluorescence nith time, AF, At, i:, automatically recorded at excitation and ernihsion wavelengths of 560 and 580 nip. From calibration plots of AF per miiiute us. concentration, the quantitj- of LAID pre.+ent in an unknown solution may be calculated. DETERMIIL'ATIOS O F -1DH. The same procedure is used as described ahove. except that 2 ml. of 0.1.11 ethyl alcohol in Tris buffer, pH 9.0, are used, and at zero time 0.1 nil. of the enzyme to be determined (containing 0.00030 t o 0.032 unit per nil. of .1DH) is added. From calibration ])lots of AF per minute 2's. ADH concentration, the amount of alcohol dehydrogenase originally lire.*ent may be determined,
+ resorufin + H20
RESULTS AND DISCUSSION
Ha0
4-
The rate of change in the fluore--( ence of the solution 151th tiine. A F minute 1proportional t o the amount of lactic 1
VOL. 36, NO. 13, DECEMBER 1964
2497
Table 11.
Dependence of AF/At upon ADH Concentration [EtOH] = 0.066M, diaphorase = 0.08 unit/ml., resazunn = 6 X 10-M AF/Al, [ADHI, umts/ml. F units/min. 0,000303 0.000606 0.00151 0 . 00606 0,0:303 0.0fioti
0.om 0.040 0.100 0.410 2.00 4.01
acid dehydrogenase, LAD, in concentrations as low as 0.000470 unit per ml. (Table I). This reaction was also first order with respect to diaphorase in concentrations of 0.0004 to 0.080 unit per ml. of solution. and to resazurin (10- to
lO'M). Hence the concentrations of these materials may be easily determined by this technique. Also, the rate of formation of r e s o r u h was proportional to the NAD+ concentrations in the region 10-'0 to lO'M, thus allowing an easy, direct determination of the quantity of this eoeneyme present. By the use of ethyl alcohol as substrate, the quantity of alcohol dehydrogenase as low as 0.000303 unit per ml. may be determined. Full details on the systems described herein, as well as applicittion to other systems, will be given in an article to follow. LITERATURE CITED
(1) Cshn, R. D., Kaplan, N. O., Levine,
L., Zwilling, E., Sctenee 136,962 (196'2). (2) Guilbnult, G. G., Kramer, D. N., ANAL.CHEM.36, 409 (1964).
(3) Kagi, J., Vallee, B. L., J. Bid. Chhem. 235, :X88 (1960). (4) Kramer, n. N., Guilbnult, G. G., ANAL.CHEM.35, 588 (1963). (5) Ibid., 36, 1662 (19M). (6) Lowry, 0. H., J . Bid. Chem. 173, 677 (1948). (7) Lowry, 0. H., Roberts, N. R., Chang, M., Ibid., 222, 97 (1956). ( 8 ) Lowry, 0. H., Roberta, N. R., Knpphahn, J. I., Ibid., 224,1047 (1957). (9) hlahler, H. R., Sarkar, N. K., Vernon, L. P., Alherty, R. A,, Ibid., 199, 585 (1952). (IO) Udenfriend, S., "Fluorescence Asmy in Biology and Medicine," p. 312, Academic Preaa, New York (196'2).
GEORGE G. GUILBAULT DAVIDN. KRAMER Defensive Research Division Chemical Resertroh and Development Labs. Edgewood Arsenal, Md. RECEIVEDfor review July 17, 1964. Accepted September 22, 1964.
Preparative Analog to Thin Layer Chromatography SIR: In the course of a study of the isolation and separation of sequirins ( I ) , a new preparative analog of thin layer chromatography (TLC) was developed. Self-supporting chromatographic columns, chromatosticks, were prepared from various TLC adsorbent materials. Chromatohars, chromatographic columns not encumbered by a containing envelope, were first prepared in 1951 by Miller and Kirschner (2) who pointed out many of the merits of baresurfaced columns, such as locating resolved zones and separating them by dissection. They employed special techniques to prepare, support, and develop t.heir chromatohars. The available TLC adsorbents are ideal for the preparation of self-supporting columns which can be processed in a manner much simpler than described by Miller and Kirschner.
Figure 1, Removal of o wet stick from the paper-roll (left). Protective holder made from aluminum wire, with the cotton pillow to transmit the developing fluid (right)
Silica gel sticks were prepared from
EXPERIMENTAI
a slurry of 25 grams of silica gel G (Merck, Darmstadt) and 50 ml. of water in a paper cylinder 20 mm. in diameter and 180 mm. in length.
Preparation of Chromatosticks. I n general, the chromatosticks were prepared by pouring a slurry containing suitable proportions of liquid and adsorbent into a cylinder rolled from filterpaper (Whatman No. l ) , and held together by strips of adhesive tape. The cylinder stands in a wooden holder in which the hottom plate contains it circular and perforated recess lined with filter paper. After a short draining period the moist column can he removed, dried, and activated.
After 15 minutes the wet chromatw stick can be removed and put into a supporting holder (Figure 1). The wire holder shown prevents breakage during handling. After being dried overnight at room temperature, and subsequently for 2 hours at 70" C., the stick was activated at 110' C. for 1 hour. Cellulose sticks were prepared from a mixture of 15 grams of MN-cellulose powder 300 G (Mackerey, Nagel & Co., Dueren) and 4.5 grams of silica gel G suspended in 70 ml. of water.
2498
ANALYTICAL CHEMISTRY
After 40 minutes, the wet stick can be removed and dried for 3 days at room temperature. Polyamide alone resulted in very fragile chromatosticks; therefore it was used in conjunction with silica gel also. Sixteen grams of Polyamide WOELM (M-\$'oelm-Eschwege, Germany) and 4 grams of silica gel G were mixed and suspended in a mixture of 80 ml. of methanol and 8 ml. of water. After draining for 60 minutes, the stick was removed and dried a t room temperature for three days. Separation Technique. The top of the chromatostick is carefully leveled wit,h a razor blade and the solution of the mixture to he separated is applied as spots in different patterns-e.g., diagonally, vertically, or in concentric circles with a micropipet: The volume of the solution should be sufficient to cover the top a t least three times to produce a uniform layer. Iletween applications, the layer is dried wit,h a fan. In common TLC, the width of the start line is 2 to 4 mm.-i.e., the diameter of the spots. In the chromatosticks the layer containing the mixture is less than 1 mm. t,hick; ttierefore, a very good start line is provided, leading to a sharper separation. After the mixture is applied, the sticks are carefully reversed in their holders and placed layer-end down on cotton pillows (Figure 1) cut from folded cotton fleece. The chromatost,icks, either singly or in groups, are placed in a previously saturated chromatography jar. The pillow uniformly transmits the developing fluid to the end of the stick
