The Effect of Heat on Crystals of Serum Albumin; Production of Crystals of Denatured Protein

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Abstract
When 0.5 cc. of serum albumin (horse) soln. (contg. 50 mg. of previously crystd. protein) was added to 12 cc. of a Na sulfate-acetate soln., crystallization began in less than 5 min., provided the soln. was warmed to 45[degree], and within 15 min., large, well formed, needle-shaped crystals were formed. At 25[degree] crystn. proceeded much more slowly. These crystals dissolved at once if 12 cc. of water was added. Heating the original soln. of serum albumin in Na sulfate-acetate to 60[degree], instead of 45[degree], caused a large flocculent ppt. of protein to appear almost at once. This ppt. was amorphous and if heating at 60[degree] was continued for 15 min., practically no protein dissolved when the suspension was subsequently cooled to room temp. and 12 cc. of water was added. In order to study the effect of heat on the crystals, they were separated by centrifuging and the original vol. of Na sulfate-acetate mixt. at 45[degree] was added to the crystals. In this medium the protein crystals were heated at various temps. from 60 to 100[degree] and finally in an autoclave at 115[degree], at each temp. for 15 min. At no temp. were the crystals destroyed, even at 115[degree]. The solubility of the heated albumin crystals was tested at room temp. by adding to each heated prep. 2 vols. of water. The crystals heated at 60[degree] dissolved in the course of 5-10 min. Crystals heated at temps. higher than 60[degree] did not dissolve completely, nor were they destroyed by being placed for several days in N HC1 or 95% EtOH. They dissolved at once in a saturated urea soln. The insol. crystals were as insol. as a heat-denatured, amorphous coagulum of serum albumin. Crystals could be dissolved in a 0.1 M borate buffer at pH 9.2. If to this soln. at 45[degree] was added the Na sulfate-acetate mixt. no crystals were formed. Instead all the protein immediately pptd., and this ppt. did not dissolve when the salt soln. was diluted with an equal vol. of water. The albumin dissolved by placing crystals previously heated at 80[degree] in the borate buffer had the properties of a denatured protein. Denaturation was not caused by the borate buffer, for if this buffer was added to native, unheated serum albumin, there was no difficulty in crystallizing the albumin and subsequently dissolving the crystals in water. Denaturation did not destroy the crystal pattern, but once the denatured albumin crystals were released from their confinement within the crystal by being dissolved in the pH 9.2 buffer it was impossible to replace them in the ordered pattern characteristic of a crystal of . native protein. It was possible to obtain crystals of denatured protein by denaturing a protein while it was in the cryst. state, but it did not seem possible to crystallize a denatured protein.

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