Crystallization and Preliminary Crystal Data of Porcine Pepsinogen*

Single crystals of porcine pepsinogen, suitable for x-ray diffraction studies, have been grown with lithium sulfate as the precipitant. These pepsinogen crystals were dissolved, activated, and assayed for proteolytic activity. The specific enzymic activity of the dissolved crystalline protein was nearly twice that of the commerical pepsinogen from which the crystals were grown. Incubation at pH 8 before assay demonstrated that the crystals are free of pepsin. This crystal form of pepsinogen belongs to the monoclinic space group C2 with 4 molecules in the unit cell. The unit cell dimensions are a = 104.8 +/- 0.5 A, b = 43.1 +/- 0.1 A, c = 88.4 +/- 0.3 A, and beta = 91.3 degrees.

the precipitant.
These pepsinogen crystals were dissolved, activated, and assayed for proteolytic activity. The specific enzymic activity of the dissolved crystalline protein was nearly twice that of the commercial pepsinogen from which the crystals were grown. Incubation at pH 8 before assay demonstrated that the crystals are free of pepsin. This crystal form of pepsinogen belongs to the monoclinic space group C2 with 4 molecules in the unit cell. Pepsinogen is the zymogen of the gastric acid protease pepsin.
Porcine pepsinogen is composed of a single polypeptide chain of 371 amino acid residues whose amino acid sequence is known (l-3

AND DISCUSSION
The pepsinogen from both sources crystallized at identical conditions and the resultant crystals were parallelepipeds with the same external morphology. Crystals as large as 1 x 0.3 x 0.3 mm have been obtained. Pepsinogen crystals also have been grown by equilibrating 17% pepsinogen solutions at pH 6 against 52% polyethylene glycol of molecular weight 400 in vapor diffusion experiments. Assays showed them to be pepsinogen but they are too small to be photographed.
Results of assays of potential proteolytic activity are shown in Table I. A 2-fold enhancement of the specific activity occurred upon crystallization. This is undoubtedly a consequence of purification by virtue of crystallization. Neither sample showed a significant diminution of activity after incubation at pH 8, where pepsin is denatured. Consequently, significant pepsin contamination of the crystals or the starting material did not occur. An x-ray diffraction photograph of the h0Z (Fig. 1) zone showed only a center of symmetry. A hk0 photograph had two mirror planes. Examination of hk0 and hkl zones demonstrated a systematic absence when h + k is odd. A facecentered monoclinic cell, space group C2, was chosen with dimensions a = 104.8 + 0.5 A, b = 43.1 k 0.1 A, c = 88.4 +  A3. These pepsinogen crystals reflect well to at least 2 A resolution and are stable in the x-ray beam for at least 5 days.
The choice of the number of molecules per unit cell can often be made by calculating Vu, the crystal volume per unit of protein molecular weight (22). With 4 pepsinogen molecules in the unit cell, V,,, is 2.52 AVdalton, which is near the median of the commonly observed values (22). Confirmation of this choice was obtained from a simple partition of volume per g of crystal between the protein fraction and the solvent fraction in the crystal (23). The formula used is: where pc and ps are the densities of the crystal and the solvent, f, is the weight fraction of protein in the crystal, and Ijp is the partial specific volume of the protein. The measured density of the pepsinogen crystals was 1.23 g/ml and the 52% saturated lithium sulfate density was 1.136 g/ml. A value of 0.733 ml/g was used for V,. The calculated weight fraction of protein in the crystal was 0.46, which implies 3.43 protein molecules/unit cell. Even though this treatment is approximate, it is sufficient to confirm the choice of 4 molecules/unit cell.
We will attempt to determine the phases for the pepsinogen diffraction data by using the results of the penicillopepsin structure via molecular replacement (24). However, since only 32% of the amino acids are identical in porcine pepsin and penicillopepsin, additional isomorphous replacement phasing may be required.
This combination was used to determine the structure of fetal hemoglobin (25).