Part 10 (1/2)

If one adds some Mg powder to oxyluciferin and then dilute acetic acid in successive additions as the acetic acid is used up in formation of Mg acetate, the oxyluciferin will be reduced relatively quickly. Nascent hydrogen is produced in the reaction and is no doubt the active reducing agent.

Dilute acid favors the reduction of oxyluciferin. If one saturates an oxyluciferin solution with CO_{2} or adds a little dilute acetic acid, HCl, HNO_{3} or H_{2}SO_{4}, to it, a certain amount of reduction will occur. No reduction occurs if the solution is saturated with pure hydrogen, even if allowed to stand 24 hours. The action of the acid begins when the solution of oxyluciferin, ordinarily slightly alkaline (PH = 9), is made neutral (PH = 7.1) as indicated in Table 9. The action of the acid must be on the oxyluciferin, as no luciferin or other enzymes destroyed on boiling are present.

TABLE 9

_Effect of Acid on Reduction of Oxyluciferin_

============================================================================= Solution

P_{H}

Luminescence

Remarks

with

luciferase

-------------------------------+------+------------+------------------------- 20 c.c. Oxyluciferin alone

9.01

Negative

20 c.c. Oxyluciferin + .05 c.c.

8.8

Negative

5 per cent. acetic acid

20 c.c. Oxyluciferin + .15 c.c.

7.1

Fair

5 per cent. acetic acid

20 c.c. Oxyluciferin + .30 c.c.

5.9

Good

Acid forms precipitate in 5 per cent. acetic acid

this oxyluciferin sol.

20 c.c. Oxyluciferin + .50 c.c.

Good

Acid forms precipitate in 5 per cent. acetic acid

this oxyluciferin sol.

20 c.c. Oxyluciferin + .75 c.c.

Good [10]

Acid forms precipitate in 5 per cent. acetic acid

this oxyluciferin sol.

[10] Light disappears quickly because of the effect of the acidity on the luciferase.

It is possible that the action of bacteria (which produces CO_{2}), muscle tissue (which contains lactic acid), milk (in which lactic acid may be formed by bacteria), or Mg + acid, in forming luciferin, is not the result of their reducing power but of their acidity. Fortunately we can test this matter by the use of reducing fluids which are not acid.

If they also form luciferin from oxyluciferin, a reduction must occur.

Nascent H can be generated by the action of NaOH on Al, or when finely divided Mg or Zn or Al is placed in water. With Mg the water becomes only slightly alkaline from formation of almost insoluble Mg(OH)_{2}. If we add some Al powder and dilute NaOH to an oxyluciferin solution, H is given off and luciferin is formed. As oxyluciferin cannot be formed by the addition of alkali alone we must have in this experiment a reduction of oxyluciferin in alkaline medium by the nascent H produced. Luciferin can also be formed by merely adding Al or Zn or Mg dust to an oxyluciferin solution. Methylene blue can also be readily reduced to its leuco-base by Zn dust or Al + NaOH.

Indeed, if one adds some Al or Zn or Mg powder to a solution of luciferase, light will appear whenever the solution is shaken.