2
02 J ournal of Natural Products, 1999, Vol. 62, No. 1
Notes
Sch em e 1
liquid nitrogen in a Waring blender. Centrifugation (5000g)
to remove insoluble debris resulted in a viable cell-free
extract.
Cell-F r ee E xt r a ct P r ep a r a t ion of A. com p r essa . A.
compressa was collected off the coast of Boca Raton, FL, at a
depth of -15 m. A voucher specimen has been depositied at
Florida Atlantic University. The sponge was maintained in
aerated seawater during transit to the laboratory. A 100 g
portion of the sponge was cut into ca. 1 cm pieces and added
to a blender containing liquid nitrogen with 200 mL of a Tris/
TES buffer, at pH 7.8, containing 5 mM DTT, 5 mM EDTA,
2
4-Methylenecholesterol (2a ) was incubated with the
sponge cell-free extract for 4 h and the resulting mixture
subjected to the formaldehyde assay. The assay resulted
in an increase in fluorescence at 460 nm of 0.5, correspond-
ing to a 0.4 µM solution of formaldehyde (Figure 1).
A number of controls were performed to confirm that the
observed increase in fluorescence was due to an enzymatic
formation of formaldehyde during the incubation of 24-
methylenecholesterol with the crude sponge enzyme ex-
tract. First, due to the complex nature of the sponge
extract, background fluorescence levels were established.
To achieve this, the untreated sponge cell-free extract was
incubated with formaldehyde dehydrogenase, reduced glu-
tathione, and NAD. This resulted in an increase in fluo-
rescence of 0.07. Second, an incubation of 24-methylene-
cholesterol was conducted with the sponge cell-free extract
that had been heated at 100 °C for 1 h, and subsequently
this mixture was subjected to the formaldehyde assay.
Again, the observed change in fluorescence was only 0.06.
Further, to confirm that the observed increase in fluores-
cence was due to the action of formaldehyde dehydroge-
nase, the formaldehyde assay was performed with the
sponge incubation mixture in the absence of FDH, resulting
in a change in fluorescence of 0.04.
3
1
0% BSA, 100 µg leupeptin, 100 µg pepstatin A, and 1 mM
PMSF, and the mixture homogenized for 5 min. The homoge-
nate was centrifuged at 5000g for 15 min, and the supernatant
was stored at -80 °C.
In cu ba tion of 24-Meth ylen ech olester ol w ith th e Cell-
F r ee Extr a ct of A. com pr essa . The cell-free extract of A.
compressa (10 mL) was thawed and incubated with 24-
methylenecholesterol (4 mg, 0.01 mmol) in a shaker water bath
at 30 °C for 4 h. The mixture was maintained at 4 °C for 0.5
h and then subjected to the formaldehyde assay.
Assa y w ith F or m a ld eh yd e Deh yd r ogen a se. The form-
aldehyde assays were performed at 25 °C by preparing a
solution of formaldehyde dehydrogenase (50 international
2 4
units) in 0.5 mL of a solution of 33 mM Na HPO (pH 7.5), 2
mM GSH, and 1 mM NAD. Fluorescence measurements were
taken by adding 10 µL of FDH solution to 2.25 mL buffer (with
GSH and NAD) and subsequently adding 0.25 mL of test
solution (either a HCHO standard or sponge incubation
mixture). Excitation was at 350 nm, and emission was
measured at 460 nm.
The significantly greater increase in fluorescence ob-
served with the experimental conditions relative to that
of the controls demonstrates for the first time the produc-
tion of formaldehyde in sterol side-chain dealkylation. The
described assay thus provides a sensitive and selective tool
for formaldehyde detection and confirms the originally
postulated mechanism shown in Scheme 1. The assay
should prove to be of general utility in other complex
systems.
Ack n ow led gm en t. Financial support was provided by the
donors of the Petroleum Research Fund, administered by the
American Chemical Society, and is gratefully acknowledged.
Refer en ces a n d Notes
(1) Kerr, R. G.; Baker, B. J . Nat. Prod. Rep. 1991, 8, 465-497.
(2) Baker, B. J .; Kerr, R. G. In Topics in Current Chemistry; Scheuer, P.
J ., Ed.; Springer-Verlag: Berlin, 1993; Vol. 167, Chapter 1, pp 1-32.
(3) Fujimoto, Y.; Morisake, M.; Ikekawa, N. Biochemistry 1980, 19, 1065-
1
069.
4) Kerr, R. G.; Baker, B. J .; Kerr, S. L.; Djerassi, C. Tetrahedron Lett.
990, 31, 5425-5428.
(5) Kerr, R. G.; Kerr, S. L.; Malik, S.; Djerassi, C. J . Am. Chem. Soc.
(
Exp er im en ta l Section
1
Gen er a l Exp er im en ta l P r oced u r es. Formaldehyde de-
hydrogenase [EC 1.2.1.1], nicotinamide adenine dinucleotide
1
992, 114, 299-303.
(
6) Nash, T. Biochem. J . 1953, 55, 416-418.
(
NAD), reduced glutathione (GSH), dithiothreitol (DTT), eth-
ylenediamine tetraacetic acid (EDTA), bovine serum albumin
BSA), leupeptin, pepstatin A, and phenylmethylsulfonyl
(
7) Sawicki, E.; Sawicki, C. R. AldehydessPhotometric Analysis; Aca-
demic Press: New York, 1975; Vol. 1, pp 210-215.
(
(8) Uotila, L.; Koivusalo, M. In Methods in Enzymology; Academic
Press: New York, 1981; Vol. 77, pp 314-320.
fluoride (PMSF) were purchased from Sigma Chemical Co.
Fluorescence measurements were recorded on a Perkin-Elmer
LS50 spectrometer.
(9) Kerr, R. G.; Kelly, K. Unpublished observations.
NP980366D