My lab and I have been attempting to get our chromogenic substrates into solution. I've read a couple papers that have used the same chromogenic substrate, Z-Gly-Pro-pNA, and they have all used methanol to dissolve it. Our lab has tried making a 100 mM Z-Gly-Pro-pNA in methanol, and it does not fully go into solution. We've tried dissolving it at 30 and 37 C. The substrate does not dissolve at 30 C and only partially at 37 C. The assay we're making this solution for is done at 37 C, so I don't think increasing the temperature past 37 C would be useful.
Try PBS:water:dioxane (8: 1.2 : 0.8 by volume)
Reference:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1134072/pdf/bj3830311.pdf
According to Sigma, it is soluble at 20 mg/ml (34 mM) in methanol, presumably at room temperature.
http://www.sigmaaldrich.com/catalog/DataSheetPage.do?brandKey=SIGMA&symbol=C2276
Unless you were planning to run the reaction in 100% methanol, the temperature at which you dissolve the compound in methanol isn't relevant. Consider what the highest methanol concentration your assay can tolerate and what the final substrate concentration needs to be. You don't need to make the stock solution any more concentrated than necessary to get that substrate concentration and that % methanol.
Have you tried DMSO? Many enzymes can tolerate it at several %.
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