Effect of Reductive Lactosamination on the Hepatic Uptake of Bovine Pancreatic Ribonuclease A Dimer*

Lactose has been coupled to the lysine residues of the cross-linked dimer of bovine pancreatic ribonuclease A by reductive amination with cyanoborohydride. Derivatives of ribonuclease dimer that contained up to 10 N’-l-(l-deoxy-lactitolyl)-lysine residues per molecule had greater than 75% of the enzymic activity of the unmodified enzyme toward yeast RNA. Upon intravenous injection of the IT-labeled (enzymically inactivated by W-carboxymethyla- tion) derivatives into rats, their uptake by the liver was a function of the number of lactose residues coupled. At 10 min, 69% of the injected derivative of ribonuclease dimer containing eight 1-deoxylactitolyl-lysine residues/molecule was found in the liver; with the non-glycosylated enzyme, the liver uptake at 10 min was only 4%, and 75% of the radioactivity was found in the kidneys. Studies on the cytostatic properties of a cross-linked


SUMMARY
Lactose has been coupled to the lysine residues of the cross-linked dimer of bovine pancreatic ribonuclease A by reductive amination with cyanoborohydride. Derivatives of ribonuclease dimer that contained up to 10 N'-l-(l-deoxylactitolyl)-lysine residues per molecule had greater than 75% of the enzymic activity of the unmodified enzyme toward yeast RNA. Upon intravenous injection of the ITlabeled (enzymically inactivated by W-carboxymethylation) derivatives into rats, their uptake by the liver was a function of the number of lactose residues coupled. At 10 min, 69% of the injected derivative of ribonuclease dimer containing eight 1-deoxylactitolyl-lysine residues/molecule was found in the liver; with the non-glycosylated enzyme, the liver uptake at 10 min was only 4%, and 75% of the radioactivity was found in the kidneys. since the secondary amine formed by reductive amination is acid-stable, only galactose is produced during the hydrolysis. The number of modified lysine residues was determined by amino acid analysis after hydrolysis in 6 N HCl, at 110" for 20 h. Nf-l-(1.deoxysorbitolyl)-lysine and N'-I-(1-deoxylactitolyl)-lysine were synthesized from a-t-BOC-L-lysine by reductive amination. On a Durrum D-500 analyzer, using pH 6 as the third buffer, unhydrolyzed N'-1-(l-deoxysorbitolyl)-lysine was eluted as a single peak located between phenylalanine and histidine. During acid hydrolysis of this derivative, three additional ninhydrin-positive peaks were produced similar to those described by Finot et al. (16) and Marsh et al. (71,although in contrast to the results of the latter authors, we did not detect the formation of any free lysine. The same products were obtained upon hydrolysis of N'-1-(l-deoxylactitolyl)-lysine. Permafluor V was the scintillation fluid), and the radioactivity was determined. The recovery of counts after oxidation, as checked by a 14C standard, was greater than 97%. The blood volume of each rat was calculated as 64.1 ml/kg of body weight (19). In experiments with derivatives of serum albumin, after killing the rat, the livers were perfused before being removed; 0.15 M NaCl was forced into the portal vein until the color of the liver became pale brown.

AND DISCUSSION
With a large excess of lactose and cyanoborohydride in the reaction medium, at pH 7 and 37", increasing amounts of lactose were coupled to RNase dimer as a function of time (Fig. 1). Glycosylated derivatives of serum albumin were similarly prepared; up to 20 mol of lactose/m01 of protein were coupled during 5 days of reaction. Although we have routinely carried out the glycosamination reaction at pH 7, subsequent experiments have shown that the coupling reaction is 2 to 3 times as fast at pH 9, a finding which is similar to the recent results of Baues and Gray (6). Amino acid analysis of the glycosylated proteins gave the same characteristic peaks observed when iV<-l-(l-deoxysorbitolyl)-lysine was hydrolyzed, which shows that the derivatives contained 1-deoxylactitolyl residues linked to the e-amino groups of lysine residues. There was generally good agreement between the results of the sugar analysis and amino acid analysis; it is, however, likely that there was some modification of the a-NH, groups of the NH,-terminal lysine residues on the RNase dimer. Derivatives of RNase dimer which contained up to 5 ldeoxylactitolyl-lysine residues/molecule retained complete activity toward cyclic 2',3'-cytidylic acid and yeast RNA. The coupling of additional lactose residues resulted in a gradual loss of activity toward both substrates (Table I), the drop in activity being most pronounced when greater than 10 lactose   No significant amounts of radioactivity were detected in the lungs, spleen, testes, and brain when these tissues were removed 10 min or 24 h after injection of RNase dimer or its glycosylated derivative.
Hepatic Uptake of Lactosaminated RNase A Dimer residues had been coupled. The activity toward poly(A) . poly(U) was, however, markedly affected by glycosylation; the coupling of 2 and 6 lactose residues/molecule of dimer resulted in 42 and 80% loss of activity, respectively.
The activity of dimeric ribonucleases toward double-stranded RNA is related to the number and most probably the exact positioning of basic charges on these enzymes (20).
After intravenous injection of [14C1RNase dimer into rats ( Fig. 2A), the protein was rapidly removed from the blood stream (half-life co. 10 min (cr Ref. 1)) and the bulk of the radioactivity at 10 min was found in the kidneys; the liver content was only 4%. In contrast, after injection of a [14ClRNase dimer derivative containing 8 l-deoxylactitolyllysine residues/molecule (Fig. 2B), the bulk of the radioactivity was found in the liver; the remaining counts were found in the kidneys. No significant amounts of radioactivity were detected in other tissues examined. Maximum hepatic uptake (69% of the total injected counts) occurred 10 min after injection; thereafter there was a gradual loss of radioactivity from the liver, which most probably reflects degradation of the protein. At 24 h after injection, less than 5% of the total radioactivity remained in the liver, while approximately 90% of the counts were found in the collected urine. When 5 mg of asialofetuin was injected with the glycosylated [V]RNase dimer derivative, only 6.5% of the total radioactivity was found in the liver 10 min after injection, which demonstrated competition for the galactose-specific receptor protein of the parenchymal cells (3).
The amounts of glycosylated RNase dimer and glycosylated serum albumin taken up by the liver were related to the number of lactose residues coupled (Fig. 3). Relatively few terminal n-galactopyranosyl residues are required for efficient plasma clearance and hepatic uptake of asialoglycoproteins (cf. Ref. 3). The present results show that 8 to 10 residues of lactose/molecule of protein are needed to give greater than 50% uptake by the liver. If the data for mole of lactose per g of protein basis, rather than per mol of protein, serum albumin is found to require fewer lactose residues to yield a given hepatic uptake than RNase dimer, probably since the albumin is not subject to the same degree of renal excretion. These results are consistent with those of Marsh et al. (7) on the effect of the number of lactose residues on the plasma clearance of asparaginase and those of Krantz et al. (21) who showed that the affinity for liver membranes of proteins that contain I-thio-P-n-galactopyranosyl residues increased with increasing numbers of coupled thiogalactoside residues. The present results show that reductive lactosamination provides a simple method for directing a protein of possible therapeutic interest to the liver even when the protein is of sufficiently low molecular weight to be subject to rapid renal clearance.