Question

An undergraduate researcher you know is confused. They were told to measure the amount of helix in poly-L-Glutamate in 0.1 M phosphate. When they made the solution using H3PO4 and KH2PO4 they saw a nice CD signal indicative of alpha helix. The next week they tried again using identical reagents except that they were out of the potassium salt and had to substitute Na2HPO4. When they made a CD measurement they saw a large negative signal at 195 nm indicating random coil conformation. How would you explain the behavior of poly-Glu to the confused student so that it made sense? What simple step did the student likely forget while making up the solution?

Answer 1

The possible reason can be to that he forgot to manitain the pH below 6 while preparing the Phosphate Buffer,

Or by any reason the pH raised above 6 by contamination.

Polyglutamic acid has a alpha helix conformation below pH 6.0 and random coil confirmation above pH 6.

Above pH 6.0, the COOH groups are converted to COO^- groups- the negative charges repel each other, disrupting the compact alpha helix and converting it to a random coil (a large negative signal at 195 nm).

The Use of Na₂HPO₄ in place of KH₂PO₄ will not have any impact on the CD measurement beacuse both will form H₃PO_4/H₂PO₄^- (Phosphate Buffer) which is prefered for CD spectra.