Hello. I work with bacterial spores. Currently, I am reading an old project report of a student who performed protein digestion from spores and estimated the amount of dityrosine (dT) present in his sample. I will first give some details and then ask my question.

(A) It is suggested that in the outermost protein layer of spores there are dityrosine (dT) cross-links present and it is estimated that every 60kDa protein from the spore outer layer has 3 dityrosines present (Pandey and Aronson 1979; Properties of the Bacillus subtilis Spore Coat).

(B) The student's (who's report I am reading) estimation was based on fluorescence measurements. dT can be detected at pH 2 with ex. 283 nm and em. 410 nm or at pH 9.5 with ex. 315 nm and em. 422 nm. For this measurement he used in vitro synthesized dT (Malencik et al., 1996; Dityrosine: Preparation, Isolation, and Analysis) with absorbance of 1.45 and ε (318 nm) = 8.7 mM-1· cm-1. From these parameters it was estimated that ~0.167mM dT was synthesized and it was used as standard for fluorescence measurement. The theoretical maximum of enzyme-catalyzed (in vitro) conversion of tyrosine to dityrosine with the above described method is ~ 25 % i.e. 0.25 mM or 250 nmol in 1 ml.

(C) The extracted protein amount that was used by the student for dT estimation was approx. 78-100 ug.

(D) The student mentions that "based on the fact that 60kDa has 3 dT, the fluorescence of 100x diluted dT standard would be comparable with that of the 2x diluted protein extract sample".

My question is, can someone explain to me how point (D) is valid?

I am attaching the papers and the extract of student's report. I hope some one can help me.

Greetings,

Wishwas.