Covalent cross-linking of proteins without chemical reagents

Abstract

A facile method for the formation of zero-length covalent cross-links between protein molecules in the lyophilized state without the use of chemical reagents has been developed. The cross-linking process is performed by simply sealing lyophilized protein under vacuum in a glass vessel and heating at 85 degrees C for 24 h. Under these conditions, approximately one-third of the total protein present becomes cross-linked, and dimer is the major product. Chemical and mass spectroscopic evidence obtained shows that zero-length cross-links are formed as a result of the condensation of interacting ammonium and carboxylate groups to form amide bonds between adjacent molecules. For the protein examined in the most detail, RNase A, the cross-linked dimer has only one amide cross-link and retains the enzymatic activity of the monomer. The in vacuo cross-linking procedure appears to be general in its applicability because five different proteins tested gave substantial cross-linking, and co-lyophilization of lysozyme and RNase A also gave a heterogeneous covalently cross-linked dimer.

Fig. 1.

SDS-PAGE of successive cycles of solubilization of 10 mg of RNase A at pH 7.0, lyophilization, then heating in vacuo for 24 hr. Total protein load per lane is 20 μg. Lane 1: low range molecular weight marker; lane 2: lyophilized RNase A, no heating in vacuo; lane 3: Cycle 1; lane 4: Cycle 2; lane 5: Cycle 3; lane 6: Cycle 4; lane 7: lyophilized RNase A heated continuously 96 h in vacuo with only one reconstitution.

Fig. 2.

SDS-PAGE of successive cycles of solubilization of 10 mg of lysozyme at pH 7.0, lyophilization, then heating in vacuo for 24 hr. Total protein load per lane is 20 μg. Lane 1: low range molecular weight marker; lane 2: lyophilized lysozyme, no heating in vacuo; lane 3: Cycle 1; lane 4: Cycle 2; lane 5: Cycle 3; lane 6: Cycle 4; lane 7: lyophilized lysozyme heated continuously 96 h in vacuo with only one reconstitution.

Fig. 3.

Size-exclusion fast performance liquid chromatography (FPLC) of in vacuo cross-linked RNase A. Separation of RNase A cross-linked products achieved with two Superdex G75 HR 10/30 columns in tandem using a mobile phase of 0.2 M Na₂HPO₄ and 0.15 M NaCl at pH 6.55. (A) RNase A lyophilized at pH 7.0, no cross-linking; (B) RNase A lyophilized at pH 7.0, then cross-linked in vacuo.

Fig. 4.

The effect of pH on the extent of RNase A dimerization. RNase A samples of 10 mg/mL were adjusted to pH values 3.0 to 10.0 with 1.0 N NaOH, lyophilized, then cross-linked under vacuum. A 10-μg sample of the treated protein was subjected to SDS-PAGE. Lane 1: RNase A at pH 3; lane 2: RNase A at pH 4; lane 3: RNase A at pH 5; lane 4: RNase A at pH 6; lane 5: RNase A at pH 7; lane 6: RNase A at pH 8; lane 7: RNase A at pH 9; lane 8: RNase A at pH 10.

Fig. 5.

Cross-linking of RNase A in the presence of trehalose at pH 7.0. RNase A (10 mg) was co-lyophilized with different amounts of trehalose and heated under vacuum for 96 h. A 20-μg sample of the treated protein was subjected to SDS-PAGE. Lane 1: RNase A alone cross-linked in vacuo; lane 2: RNase A and trehalose in a 5 : 1 (w/w) ratio, cross-linked in vacuo; lane 3: RNase A and trehalose in a 1 : 1 (w/w) ratio, cross-linked in vacuo; lane 4: RNase A and trehalose in a 1 : 5 (w/w) ratio, cross-linked in vacuo.

Fig. 6.

Heterogeneous cross-linking of RNase A and lysozyme. RNase A (10 mg) was co-lyophilized with lysozyme (10 mg) at pH 7.0, heated under vacuum for 48 hr, and 15 μg of the treated protein was subjected to SDS-PAGE. Lane 1: RNase A (pH 7.0) alone cross-linked in vacuo, 48 h; lane 2: lysozyme (pH 7.0) alone, cross-linked in vacuo, 48 h; lane 3: RNase A (pH 7.0) and lysozyme (pH 7.0) co-lyophilized and cross-linked in vacuo, 48 h. The lysozyme-RNase A dimer is indicated by the arrow.

Fig. 7.

SDS-PAGE showing the effect of chemical modification of RNase A (15 mg/mL) on in vacuo cross-linking. Total protein loaded per lane is 10 μg. (A) Lane 1: RNase A lyophilized at pH 9.0 with no in vacuo treatment; lane 2: RNase A lyophilized at pH 9.0 and heated in vacuo, 48 h; lane 3: reductively methylated RNase A lyophilized at pH 9.0, and heated in vacuo, 48 h. (B) Lane 1: RNase A with amidated carboxyl groups lyophilized at pH 7.0 and heated in vacuo, 48 h.

Fig. 8.

Deconvoluted electrospray mass spectrum of the RNase A dimer produced by in vacuo cross-linking.