J. Mol. Biol. (1966) 15, 683-684

The Unit Cell of Crystalline ex-Lactalbumin

Several methods have been described for the isolation and crystallization ofe-lactalbumin from cow's milk (Zweig & Block, 1954; Gordon & Ziegler, 1955a;Aschaffenburg & Drewry, 1957; Gordon, 1964; Robbins & Kronman, 1964). In eachinstance crystallization was achieved with salting-out concentrations of ammoniumsulfate near pH 6·6. Lactalbumin forms amorphous precipitates from water solu­tions near pH 4'0, and to our knowledge no one has produced crystals of the proteinfrom low ionic strength solutions.

Crystallized IX-lactalbumin has a molecular weight in the range of 14,900 to 16,500(Gordon & Semmett, 1953; Gordon & Ziegler, 1955b; Wetlaufer, 1961). The moleculepossesses one N- and one C-terminal group (Yasunobu & Wilcox, 1958; Wetlaufer,1961; Weil & Seibles, 1961) and apparently consists of a single polypeptide chain.

In this study IX-lactalbumin was isolated, following the method of Robbins &Kronman (1964), and purified by crystallizing five times from ammonium sulfatesolutions. The crystals used in taking the X-ray photographs and in making the densitymeasurements were grown from a solution initially adjusted to 51% saturation withammonium sulfate, with 1-56% (w/v) protein, at pH 6·64. The solution was allowedto stand for many months in a stoppered vial in a refrigerator. Crystals formed whichwere large enough (up to about 0·35 mm X 0·5 mm X 1·0 mm) for single-crystaldiffraction photography. They appeared as elongated parallelepipeds which wereoccasionally arranged in rosettes. Other shapes have been reported (Aschaffenburg &Drewry, 1957) for crystals obtained under essentially the same conditions. The variousshapes probably represent different habits of the same crystal form.

The material we examined had the symmetry of space group A2. The values listedfor the cell constants were averaged from hkO, hOl and Okl precession photographs,and are, for the face-centered monoclinic cell:

a = 94-8 ± 0-3, b = 122·4 ± 0'3, C = 117·8 ± 0·5 A,

f3 = 116° 13' ± 9', U = 1·226 X 106 As.

The protein content of the crystals (p; grams of dry-weight protein per ems of wetcrystals) was approximated by the relationship

• Pc - PsPp = -

1 - v'Ps

where Pc is the measured density of the wet crystal, Ps the measured density of themother liquor (minus the small, calculated density contribution of dissolved protein),and v the reported partial specific volume of IX-lactalbumin, 0-729 cm3/g, calculatedfrom its amino acid composition (Gordon & Ziegler, 1955b). Densities of wet crystalswere determined by the flotation method using mixtures of chlorob enzene and bromo­benzene which had been equilibrated with an ammonium sulfate solution of approxi­mately the same concentration as the mother liquor. Values of 1·213 and 1·210 g/cms

(22°C) were obtained for material from two separate vials . The corresponding Ps

values were 1-156 and 1·155 g/cm s (22°0).683

684 J. K. INMAN AND R. F. BRYAN

The above expression for p; assumes that Ps and v will reasonably approximateconditions within the crystal. This expression provides a convenient estimate ofprotein content in cases where crystals are too small to allow a determination bydirect weighing and protein assay of individual crystals. Accordingly, we obtained anaverage value for p; of 0·357 gjcm3 of wet crystal, from which one can determine asum of atomic weights, LA, for the asymmetric unit:

"" U.~A = 4" . pp. N = 65,900

where U is the volume of the unit cell and N is Avogadro's number. From the re­ported values of the molecular weight of the protein, it appears that the asymmetricunit contains four molecules of ex-lactalbumin, and that the corresponding face­centered unit cell contains 16 molecules. None of the photographs taken providesobvious evidence of symmetry within the asymmetric unit with respect to the arrange­ment of the polypeptide chains.

Department of BiophysicsJohns Hopkins UniversitySchool of MedicineBaltimore 5, Maryland, U.S.A.

Received 13 December 1965

JOHN K. INMAN

ROBERT F. BRYAN

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