1
276 John T. Reilly et al.
The photochemical reactions under conditions in which
levels of histidine, trans-UCA, cis-UCA and HAL activity,
comparing subjects undergoing PUVA therapy with those that
are not, is needed. Third, a study to determine the type of
photochemical reaction responsible for the inhibition of HAL,
in vitro, is necessary.
oxygen is present and absent from the sample being irradiated
will have to be investigated in the future. It is generally
accepted that there are three types of reactions of photosen-
sitization (33,34). In Type I reactions, the excited state of
psoralen can generates radicals in the substrate as shown:
3Psoralen + Substrate ! Psoralen + Substrateꢀ
CONCLUSIONS
Presented in this paper were the effects that 8-methoxyp-
soralen and POPs have on the catalysis of histidine to trans-
urocanic acid by HAL (EC 4.3.1.3) using an enzymatic assay
that monitored the growth of the trans-urocanic acid peak at
In turn, the substrate may take up oxygen. In Type II
reactions, the excited state of psoralen transfers its energy to
molecular oxygen generating singlet oxygen; in turn, singlet
oxygen can react with the histidase, psoralen or other species
present as shown:
2
77 nm. The dark binding experiments of HAL have shown
there is no inhibition due to denatured ethanol and the type
of inhibition caused by 8-methoxypsoralen was determined
3
1
Psoralen + O2 ! O2 þ Psoralen
to be of
a noncompetitive type. POPs, formed when
In Type III reactions the excited state of psoralen reacts
directly with the substrate forming a covalently bonded
photoadduct as shown:
irradiated by both broadband UVA and broadband UVA ⁄
UVB, were found to inhibit HAL. HAL, when irradiated
with filtered radiation (322–400 nm), was not destroyed due
to photo-oxidation of the amino acids but did show
inhibition when compared to the baseline. When unfiltered
irradiation (300–400 nm) was used, the enzyme was found to
be inhibited more than when irradiated with UVA radiation
alone.
3
Psoralen + Substrate ! Substrate ꢁ Psoralen
When a solution is deoxygenated, Type I and II reactions can
be distinguished from Type III reactions. If we eliminate
oxygen, different POPs are obtained and we would expect
different inhibition. If Type I and II reactions are eliminated
as the source of inhibition, this research takes on a new
dimension. Similar to what Sastry (30) accomplished, an
examination of the possible photoproducts would have to be
undertaken. If Type III is eliminated we will have to investigate
the role that oxygen and singlet oxygen play in the inhibition of
HAL. The specific type of photochemical reaction will be
investigated in the future.
As mentioned above, the long-range goal of this research is
to determine the role that PUVA therapy has on the enzyme
HAL and its role in photoimmunosuppression. If PUVA
therapy is shown to inhibit HAL activity in vivo, theoretically
less trans-UCA will be produced and, in turn, less cis-UCA.
The question then becomes, does the suppression of trans-
UCA acting as a natural photoreceptor, and cis-UCA, acting
as an immunosuppressant, affect the immuno-suppression of
patients undergoing PUVA therapy? Vestey and Norval (35)
showed that there was an anti-psoriatic effect in subjects with a
topical application of cis-UCA which, again, suggest that these
two modes (PUVA therapy and cis-UCA) of photoimmuno-
suppression are not causally linked. In addition, what role, if
any, does this have on the more serious side effects of PUVA
therapy, the increased risk of squamous cell carcinoma and
melanoma (2–4)? DeFabo et al. (25) addresses part of this
issue in their paper on levels of trans-UCA in mice regulated
by dietary levels of l-histidine and their relevance to cancer.
Their roles are still under investigation.
Acknowledgements—The authors would like to thank Janus Retey
and colleagues from the University of Karlsruhe, Institute of Organic
Chemistry for their generous help in obtaining the modified Escher-
ichia coli. The author, J.R., would like to thank NSF ⁄ RSEC
Fellowship, Grant CHE9974734, for their generous support. The
authors would like to thank Coastal Carolina University and Elon
University, Departments of Chemistry for their support. The authors
would like to thank Michael Pierce from the Department of Biology,
Coastal Carolina University for his help in harvesting and purifying
HAL.
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