The Influence of Aldehyde Chain Length upon the

THE JOURNAL

OB BIOLOGICAL

CHEMISTRY

Vol. 238, No. 9, September 1963 Printed

in

U.S.A.

The Influence Quantum J.

of Aldehyde Chain Length Yield of the Bioluminescent Achromobacter fischeri* WOODLAND

From the Division

HASTINGS,

JAMES

SPUDICH,

for publication,

research

has been

MALNIC~

supported

part by a contract

in

from

Office of Naval Research and bv a nrant from the National Science Foundation. Permanent address, t Fellow of the Rockefeller Foundation. Department of Physiology, Faculty of Medicine, University of Siio Paulo, S&o Paulo, Brazil. I

-

and the Marine

April 8, 1963)

mental conditions) from a few secondsto a major fraction of an hour. As indicated in the hypothetical reaction schemein Fig. 1, the intermediate has two possiblefates. In the absenceof aldehyde the intermediate decays with a very low light emission; this is referred to as the endogenouslight emission. However, if aldehyde

is present,

a far greater

amount

of luminescence

is

obtained from the samequantity of enzyme intermediate. The effect of aldehyde upon the reaction may therefore be described asan effect upon the quantum yield per moleof the enzymeintermediate. The aldehydestested have beenfound to differ with respectto the quantum yield experimentally measured. EXPERIMENTAL

PROCEDURE

Several of the aldehydesusedin theseexperiments,as well as the fatty acids, wereobtained from commercialsources(Matheson, Coleman,and Bell, East Rutherford, New Jersey, Aldrich ChemicalCompany, Milwaukee, and Eastman Kodak, Rochester, New York). Those aldehydesnot commerciallyavailable were synthesized from the correspondingfatty acid by the LiAlH, reduction procedure recently reported by Brown and Tsukamoto (9). First, SOClzwasusedto makethe acid chloride. After a vacuum distillation of the product, aziridine (ethylenimine) wasusedto form the acylaziridine, and the HCl formed was precipitated with triethylamine. The (C&H&N-HCl was filtered off and the acylaziridine wasreducedwith a 1.25M LiAlH ether solution. Anhydrous ether was usedas the solvent throughout the preparation. All the reactions except the synthesis of the acid chloride were carried out at lessthan 0” in an ice-saltbath. Subsequentto the reduction, the ether was removed with a water aspirator, and the aldehyde was vacuum-distilled. A final purification wasachieved by collection from a gas chromatography unit by use of an Aerograph (Wilkins Instrument Company, Walnut Creek, California) dual columngaschromatograph. The column (5 feet X 0.25 inch) consistedof 20% dichlorodimethylsilane on Celite 545. The temperatures and flow rates wereadjustedto give goodseparationof the impurities from the various aldehydes. Larger quantities of pure aldehydes were obtained with an -4erographpreparative model gas chromatograph. Saturated aqueoussolutionsof aldehyde at 25” were prepared by first mixing excesspure aldehyde with water. Equilibrium

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A long chain saturated fatty aldehyde is required for maximal light emission in the bioluminescent reaction catalyzed by the enzyme, bacterial luciferase, purified from extracts of Achromobatter &ch.t&. Since the discovery of this requirement by Strehler and Cormier (1, 2), very little has been learned about either the chemical role of aldehyde in the reaction or the nature of the presumed naturally occurring aldehyde-like compound. Indeed, it is not yet definitely known whether aldehyde is actually consumed in the reaction, as suggested by the experiments of McElroy and Green (3) and of Cormier and Totter (4), or whether it has instead a catalytic-like role, as suggested by Strehler (5). With respect to the specificity of the reaction, Strehler and Cormier (2) showed that several different aldehydes stimulate light emission, albeit with varying degrees of effectiveness as judged by the initial maximal intensity (initial rate) of the reaction. Additional studies with dark mutants of the luminous bacteria, as well as the reaction in vitro with partially purified enzyme, were reported by Rogers and McElroy (6). In view of the increased knowledge about the mechanism of the bioluminescent reaction in bacteria (7, 8), information concerning the effects of different aldehydes is of considerable interest. In the present study, homologous saturated straight chain aliphatic aldehydes ranging from 3 to 20 carbon atoms were examined with regard to their effect upon the reaction. The chemiluminescent quantum yield of the reaction has been found to be different with aldehydes of different chain length. This effect is believed to be localized at a step involved in either the creation or dissipation of the excited state. The reaction requires, in addition to the aldehyde and enzyme, reduced flavin mononucleotide and oxygen. It has been recently shown that the light-emitting reaction involves first the reduction of enzyme by reduced flavin mononucleotide, followed by the combination of reduced enzyme with oxygen (Fig. 1). Added aldehyde is not required for these reactions, and the enzyme intermediate resulting from these two steps has an appreciable lifetime which may vary (depending upon experi* This

GERHARD

of Biochemistry, University of Illinois, Urbana, Illinois, Biological Laboratory, Woods Hole, Massachusetts (Received

the

AND

upon the Relative Reaction of

J. W. Hastings, J. SpwEich, and G. Malnic

September 1963

Enr

t

:;

FMNH,

‘*

FMN

l

+ H,OL

‘5

En2

t FMN

LOW QVANTUM YIELD REACTION

SH Em

’ \

I

-Enz